A tumor vaccine and its preparation method and application

Abstract

A tumor vaccine, its preparation method, and application. Specifically, it relates to a drug combination comprising a first membrane component comprising a membrane derived from the inner membrane of bacteria, the drug combination further comprising a component derived from an organism other than the bacteria, and a preparation method and application of the drug combination.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a tumor vaccine and its preparation method and application. Background Art

[0002] Currently, surgery remains the preferred treatment option for cancer, playing a crucial role in cancer diagnosis, staging, reconstruction, and palliation. Advances in traditional chemoradiotherapy and novel adjuvant chemoradiotherapy have significantly improved the prognosis of cancer patients. However, despite continuous advancements in treatment techniques, local tumor recurrence and metastasis remain major sources of morbidity and mortality in cancer patients. Numerous studies have found that surgical manipulation and damage to tumor margins may cause individual tumor cells to shed into the peripheral circulation. Furthermore, surgical stress can affect neuroendocrine regulation, metabolism, inflammatory responses, and the immune microenvironment surrounding tumor cells, leading to immunosuppression. Following major surgery, cellular immunosuppression can persist for several days, creating a window of opportunity for tumor invasion of the immune system. This period of immunosuppression is often manifested by a loss of tumor cell surveillance and a decrease in peripheral blood NK cells, cytotoxic T lymphocytes, dendritic cells, and helper T lymphocytes.

[0003] Currently, the types of vaccines used to prevent postoperative tumor recurrence are relatively limited. CN104619833A describes a peptide vaccine for cancer, specifically an epitope peptide derived from UBE2T that triggers CTLs, and further provides a pharmaceutical composition containing such an epitope peptide derived from UBE2T or a polynucleotide encoding the polypeptide as an active ingredient. In addition, the invention provides a method for treating and / or preventing, and / or preventing, postoperative recurrence thereof, and a method for inducing CTLs, a method for inducing anti-tumor immunity, which uses the epitope peptide derived from UBE2T of the invention, a polynucleotide encoding the peptide, or an antigen-presenting cell presenting the peptide, or a pharmaceutical composition. CN109481418A discloses an anti-tumor nanoparticle, a preparation method and application thereof, and the anti-tumor nanoparticle provided comprises a core, a coating layer coating the core, and an immune layer coating the coating layer. The anti-tumor nanoparticles obtained by coating the nanoparticles with an immune adjuvant can achieve a combined therapeutic effect of photodynamic therapy and immunotherapy. However, the current types of vaccines for preventing postoperative tumor recurrence are limited, and the field needs vaccines with better efficacy, higher safety, easier preparation, and wider applicability. Therefore, it is of great significance to develop a new type of tumor vaccine with significant efficacy for preventing postoperative cancer recurrence and its preparation method. Summary of the Invention

[0004] In one aspect, the present application provides a pharmaceutical combination comprising a first membrane component, wherein the first membrane component comprises a membrane derived from the inner membrane of bacteria, and the pharmaceutical combination further comprises a component derived from an organism other than the bacteria.

[0005] In one embodiment, the other organism comprises a cell.

[0006] In one embodiment, the other organism comprises a mammalian cell.

[0007] In one embodiment, the other organism comprises a tumor cell.

[0008] In one embodiment, the other organism comprises solid tumor cells.

[0009] In one embodiment, the other organism is selected from the group consisting of breast cancer cells, colon cancer cells, liver cancer cells, gastric cancer cells, kidney cancer cells, pancreatic cancer cells, ovarian cancer cells, lymphoma cells, osteosarcoma cells, glioma cells, prostate cancer cells, and melanoma cells, and any combination thereof.

[0010] In one embodiment, said component of said other organism comprises an immunogenic component.

[0011] In one embodiment, said component of said other organism is capable of eliciting an immune response to said organism.

[0012] In one embodiment, said component of said other organism comprises a component derived from a cell membrane of said organism.

[0013] In one embodiment, said component of said other organism comprises a tumor antigen or a functionally active fragment thereof.

[0014] In one embodiment, the component of the other organism further comprises a protein or a functionally active fragment thereof selected from the following group: antigen peptide transporter 1, H-2 class II histocompatibility antigen γ chain, tyrosine protein kinase SYK, high affinity immunoglobulin epsilon receptor subunit γ, Ras-related C3 botulinum toxin substrate 2, tyrosine protein kinase BTK, receptor type tyrosine protein phosphatase C, Na + / K +-ATPase, ATP5A (IM CVa), ubiquinone cytochrome c reductase core protein I (IMCore I), VDAC1 / porin (OM Porin), matrix cyclophilin D (Matrix CypD), intermembrane space cytochrome c (IMSCytc), basement membrane-specific heparan sulfate proteoglycan core protein (HSPG2), plasma membrane calcium-transporting ATPase 1 (ATP2b1), endoplasmic reticulum membrane protein complex subunit 1 (EMC1), transmembrane protein 43 (TMEM43), vesicle membrane protein VIP36 (LMAN2), vesicle-associated membrane protein-associated protein A (VAPA), transmembrane 9 superfamily member 2 (TM9SF2), transmembrane emp24 domain-containing protein 10 (TMED10), adipocyte plasma membrane-associated protein (APAMP), synaptic vesicle membrane protein VAT (VAT1), nuclear pore membrane glycoprotein 210 (NUP210), plasma membrane calcium-transporting ATPase 4 (ATP2b4), plasma membrane calcium-transporting ATPase 2 (ATP2b2), erythrocyte band 7 integral membrane protein (STOM), transmembrane emp24 domain-containing protein 9 (TMED9), mitochondrial import inner membrane transposase subunit TIM44 (TIMM44), membrane-associated progesterone receptor component 1 (PGRMC1), ER membrane protein complex subunit 3 (EMC3), vesicle-associated membrane protein-associated protein B (VAPB), membrane primary amine oxidase (AOC3), vesicle-associated membrane protein 7 (VAMP7), Golgi membrane protein 4 (GOLIM4), membrane-associated progesterone receptor component 2 (PGRMC2), transmembrane 9 superfamily member 3 (TM9SF3), transmembrane 9 superfamily member 4 (TM9SF4), endoplasmic reticulum membrane protein complex subunit 2 (EMC2), mitochondrial import inner membrane transposase subunit TIM50 (TIMM50), peroxide Proteosomal membrane protein 11B (PEX11b), transmembrane emp24 domain-containing protein 2 (TMED2), secretory carrier-associated membrane protein 3 (SCAMP3), thioredoxin-related transmembrane protein 4 (TMX4), peroxisomal membrane protein PMP34 (SLC25a17), peroxisomal membrane protein PEX14 (PEX14), endoplasmic reticulum membrane protein complex subunit 8 (EMC8), interferon-induced transmembrane protein 3 (IFITM3), lysosomal-associated membrane glycoprotein 2 (LAMP2), thioredoxin-related transmembrane protein 2 (TMX2), vesicle-associated membrane protein 3 (VAMP3), lysosomal-associated membrane glycoprotein 1 (LAMP1), mitochondrial import inner membrane transposase subunit Tim8A (TIMM8a1), lysosomal membrane protein 2 (SCARB2), Iggamma-2A chain C region (IGHG2a), transmembrane 9 superfamily member 1 (TM9SF1), inner nuclear membrane protein Man1 (LEMD3), transmembrane emp24 domain-containing protein 4 (TMED4), Thy-1 membrane glycoprotein (Thy1), mitochondrial import inner membrane transposase subunit Tim23 (TIMM23), mitochondrial import inner membrane transposase subunit Tim9 (TIMM9), mitochondrial import inner membrane transposase subunit Tim13 (TIMM13), secretory carrier-associated membrane protein 2 (SCAMP2), secretory carrier-associated membrane protein 1 (SCAMP1), integral membrane protein 2B (ITM2b), mitochondrial import inner membrane transposase subunit Ti m10 (TIMM10), vacuole membrane protein 1 (VMP1), growth hormone-induced transmembrane protein (GHITM), death domain-containing membrane protein (NRADD), vesicle-associated membrane protein 8 (VAMP8), transmembrane anterior-posterior transition protein 1 (TAPT1), mitochondrial intermembrane space import and assembly protein 40 (CHCHD4), glycosylated lysosomal membrane protein (GLMP), transmembrane 4 domain superfamily A member 6D (MS4A6D), translocation chain-associated membrane protein 1 (TRAM1), interferon-induced transmembrane protein 2 (IFITM2), sarcolemma-associated protein (SLMAP), membrane magnesium transporter 1 (MMGT1), nuclear envelope pore protein POM121 (POM121), transmembrane protein C16orf54 homologous protein (AI467606), vacuolar ATPase assembly integral membrane protein Vma21 (VMA21), epithelial membrane protein 1 (EMP1), membrane-associated phosphatidylinositol transfer protein 1 (PITPNM1), small integral membrane protein 15 (SMIM15), nuclear integral membrane protein 1 (NEMP1), integral membrane protein GPR180 (GPR180), secretory carrier-associated membrane protein 4 (SCAMP4), translocation chain-associated membrane protein 2 (TRAM2), transmembrane and coiled-coil domain protein 3 (TMCC3), matrix membrane-associated protein 1 (SMAP1), neuronal membrane glycoprotein M6-b (GPM6b), epithelial membrane protein 2 (EMP2), Golgi membrane protein 1 (GOLM1), SI D1 transmembrane family member 2 (SIDT2), trans-Golgi network integral membrane protein 2 (TGOLN2), Golgi membrane protein TVP23 homolog B (FAM18b), mitochondrial inner membrane protein OXA1L (OXA1L), ossification-related transmembrane protein 1 (OSTM1), peroxisomal membrane protein PEX13 (PEX13), unilaterally passing membrane and coiled-coil domain protein 1 (SMCO1), distal membrane arm assembly complex protein 1 (DMAC1), secretory carrier-associated membrane protein 5 (Scamp5), cystic fibrosis transmembrane conductance regulator (CFTR), osteoclast-stimulating transmembrane protein (OCSTAMP), fat storage-inducing transmembrane protein 2 (FITM2) and transmembrane channel-like protein 5 (TMC5), and any combination thereof.

[0015] In one embodiment, the component of the other organism further comprises a protein or a functionally active fragment thereof selected from the following group: antigen peptide transporter 1, H-2 class II histocompatibility antigen γ chain, tyrosine protein kinase SYK, high affinity immunoglobulin epsilon receptor subunit γ, Ras-related C3 botulinum toxin substrate 2, tyrosine protein kinase BTK, receptor type tyrosine protein phosphatase C, Na + / K + -ATPase, ATP5A (IM CVa), ubiquinone cytochrome c reductase core protein I (IMCore I), VDAC1 / porin (OM Porin), matrix cyclophilin D (Matrix CypD) and intermembrane space cytochrome c (IMSCytc), and any combination thereof.

[0016] In one embodiment, it comprises a second membrane fraction comprising a membrane derived from a cell membrane of said other organism.

[0017] In one embodiment, the bacteria comprise Gram-negative bacteria and / or Gram-positive bacteria.

[0018] In one embodiment, the bacteria are selected from the group consisting of Escherichia coli, Staphylococcus, Bacillus, Lactobacillus, Klebsiella, Brucella, Proteus, Acinetobacter and Pseudomonas, and any combination thereof.

[0019] In one embodiment, the inner membrane of the bacteria comprises a protein or a functionally active fragment thereof selected from the following groups: cell division protein FtsZ, inner membrane protein YhcB, inner membrane protein translocation enzyme YidC, cell division protein NlpI, ABC transporter protein MsbA, inner membrane transport protein TatA, membrane phospholipid transport system lipoprotein MlaA, inner membrane protein TolQ, membrane transport lipoprotein PqiC, outer membrane protein TolC, outer membrane introduction protein FimD, outer membrane pore protein OmpC, membrane transport protein PqiB, major outer membrane lipoprotein Lpp, membrane-bound hemolytic wall protein transglycosylase MltB, UPF0194 membrane protein YbhG, putative membrane protein IgaA homolog, transmembrane phospholipid transport system lipoprotein Protein MlaA, inner membrane protein YejM, membrane-bound hemolytic wall protein transglycosylase MltA, transmembrane phospholipid transport system binding protein MlaC, inner membrane protein YlaC, transmembrane transport protein YebT, membrane-bound hemolytic murein transglycosylase MltC, transmembrane phospholipid transport system ATP-binding protein MlaF, intermembrane phospholipid transport system binding protein MlaD, inner membrane protein YqjE, UPF0053 inner membrane protein YfjD, UPF0053 inner membrane protein YoaE, inner membrane type lytic murein transglycosylase A, UPF0394 inner membrane protein YedE, intermembrane phospholipid transport system binding protein MlaB, inner membrane protein YccF, transmembrane phospholipid transport system permease protein MalE, inner membrane protein YedI, inner membrane protein YgaP, membrane transport protein PqiA, UPF0056 membrane protein YhcE, inner membrane protein YbhL, inner membrane protein YhjD, inner membrane transport protein YbaT, inner membrane protein YjjP, inner membrane protein YhaH, inner membrane protein YbjJ, inner membrane transport protein YqeG, UPF0053 inner membrane protein YtfL, arsenic pump membrane protein ArsB, inner membrane protein YpjD, membrane-bound lysozyme inhibitor MliC of C-type lysozyme, UPF0283 membrane protein YcjF, UPF0259 membrane protein YciC, inner membrane protein YgfX, inner membrane protein YbbJ, lipoprotein release system transmembrane protein LolE, inner membrane protein YjcH, protein export membrane protein SecG, inner Membrane protein YfdC, UPF0324 inner membrane protein YeiH, UPF0266 membrane protein YobD, TVP38 / TMEM64 family inner membrane protein YdjZ, membrane-associated protein UidC, inner membrane protein YbiR, inner membrane protein YhiM, membrane transport protein YfcA, inner membrane protein YdgK, membrane-bound hemolytic wall protein transglycosylase F, multidrug resistance outer membrane protein MdtP, UPF0208 membrane protein YfbV, peptidoglycan-associated lipoprotein, lipoprotein YiaD, lipoprotein YeaY, apolipoprotein N-acyltransferase, D-methionine-binding lipoprotein MetQ, lipoprotein GfcD, lipoprotein YbjP, lipoprotein YgeR, lipoprotein release system ATP-binding protein LolD,Osmogen-inducible lipoprotein OsmE, Osmogen-inducible lipoprotein OsmB, lipoprotein YdcL, lipoprotein YajG, lipoprotein NlpE, phosphatidylglycerol-lipoprotein diacylglycerol transferase, lipoprotein YghJ, lipoprotein YedD, lipoprotein YifL, lipoprotein YgdI, lipoprotein YbaY, lipoprotein signal peptidase, lipoprotein YceB, lipoprotein YajI and inner membrane protein YebE, and any combination thereof.

[0020] In one embodiment, the inner membrane of the bacterium comprises a protein or a functionally active fragment thereof selected from the following group: FtsZ, YhcB, YidC, NlpI, MsbA, TatA, MlaA, TolQ and YebE, and any combination thereof.

[0021] In one embodiment, the pharmaceutical composition further comprises an inner core.

[0022] In one embodiment, the inner core comprises a biocompatible material.

[0023] In one embodiment, the inner core comprises a synthetic material.

[0024] In one embodiment, the inner core comprises a substance selected from the following group: polylactic acid-co-glycolic acid (PLGA), metal-organic framework (MOF), polycaprolactone (PCL), polyamide-amine (PAMAM), carbon nanotubes, graphene, gold nanoparticles, mesoporous silica nanoparticles, iron oxide nanoparticles, silver nanoparticles, tungsten nanoparticles, manganese nanoparticles, platinum nanoparticles, quantum dots, aluminum oxide nanoparticles, hydroxyapatite nanoparticles, lipid nanoparticles (LNP), DNA nanostructures, nanohydrogels, rare earth fluoride nanocrystals and NaYF4 nanoparticles, and any combination thereof.

[0025] In one embodiment, the inner core comprises a substance selected from the group consisting of an immune adjuvant, an immune checkpoint inhibitor, a nucleic acid molecule, and a chemotherapeutic agent, and any combination thereof.

[0026] In one embodiment, the inner core comprises the immunoadjuvant monophosphoryl lipid A.

[0027] In one embodiment, the inner core comprises doxorubicin, paclitaxel, docetaxel, gemcitabine, capecitabine, cyclophosphamide, fluorouracil, pemetrexed, raltitrexed, bleomycin, daunorubicin, doxorubicin, vincristine, and etoposide, and any combination thereof.

[0028] In one embodiment, the inner core comprises an indoleamine 2,3-dioxygenase (IDO) inhibitor.

[0029] In one embodiment, the inner core comprises small interfering RNA (siRNA).

[0030] In one embodiment, the inner core has a diameter of about 60 to about 100 nanometers.

[0031] In one embodiment, the inner core has a diameter of about 86 nanometers.

[0032] In one embodiment, the pharmaceutical combination comprises a shell comprising the first membrane component and the second membrane component.

[0033] In one embodiment, the shell comprises a fused membrane of the first membrane component and the second membrane component.

[0034] In one embodiment, the shell has a thickness of about 10 to about 20 nanometers.

[0035] In one embodiment, the shell has a thickness of about 14 nanometers.

[0036] In one embodiment, the shell has a diameter of about 100 nanometers.

[0037] In one embodiment, the surface potential (Zeta potential) of the shell is from about +50 mV to about -50 mV.

[0038] In one embodiment, the surface potential (Zeta potential) of the shell is about -21 mV.

[0039] In one embodiment, the mass ratio of the first membrane component to the second membrane component in the pharmaceutical combination is 1:100 to 100:1.

[0040] In one embodiment, the mass ratio of the first membrane component to the second membrane component in the drug combination is 1:100, 1:75, 1:50, 1:25, 1:10, 1:5, 1:3, 1:1, 1:0, 0:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1 or 100:1.

[0041] In one embodiment, the presence and / or ratio of the first membrane fraction to the second membrane fraction in the pharmaceutical combination is confirmed by Western blotting and / or immunogold staining.

[0042] In one embodiment, the pharmaceutical composition comprises particles comprising the inner core and the outer shell.

[0043] In one embodiment, the mass ratio of the shell to the core material is from about 1:1 to about 1:10.

[0044] In one embodiment, the mass ratio of the shell to the core material is from about 1:4 to about 1:6.

[0045] In one embodiment, the lipopolysaccharide (LPS) content of the particles is not significantly different from the LPS content of mammalian cells.

[0046] In one embodiment, the particles have a diameter of about 70 to about 120 nanometers.

[0047] In one embodiment, the particles are about 100 nanometers in diameter.

[0048] In one embodiment, the diameter is measured by transmission electron microscopy (TEM).

[0049] In one embodiment, the particles have a hydrated particle size of about 150 nanometers to about 250 nanometers.

[0050] In one embodiment, the particles have a hydrated particle size of about 180 nanometers.

[0051] In one embodiment, the surface potential (Zeta potential) of the particles is from about +50 mV to about -50 mV.

[0052] In one embodiment, the surface potential (Zeta potential) of the particles is about -21 mV.

[0053] In one embodiment, the hydrated particle size and / or surface potential of the particles are measured by a dynamic light scattering (DLS) instrument.

[0054] In one embodiment, the particles are capable of remaining stable in solution.

[0055] In one embodiment, the stability comprises that the hydrated particle size and / or surface potential of the particles after being stored for a period of time are not significantly different from those before the period of time.

[0056] In one embodiment, the stability comprises that the hydrated particle size and / or surface potential of the particles after being stored in phosphate buffered saline (PBS) at 4 degrees Celsius for a period of time are not significantly different from those before the period of time.

[0057] In one embodiment, the period of time is no less than about 21 days.

[0058] In one embodiment, it further comprises a substance selected from the following group: an immune adjuvant, an immune checkpoint inhibitor, a nucleic acid molecule, a chemotherapeutic agent and a photosensitizer, and any combination thereof.

[0059] In one embodiment, it further comprises the immunoadjuvant monophosphoryl lipid A (MPLA).

[0060] In one embodiment, it further comprises an indoleamine 2,3-dioxygenase (IDO) inhibitor.

[0061] In one embodiment, it further comprises small interfering RNA (siRNA).

[0062] On the other hand, the present application also provides a vaccine comprising the drug combination of the present application.

[0063] On the other hand, the present application also provides a kit comprising the drug combination and / or the vaccine of the present application.

[0064] On the other hand, the present application also provides a method for enhancing the uptake of a target antigen by immune cells, comprising providing a drug combination comprising a first membrane component comprising a membrane derived from the inner membrane of bacteria, and the drug combination further comprising the target antigen.

[0065] In one embodiment, the pharmaceutical combination comprises the pharmaceutical combination of the present application.

[0066] In one embodiment, the immune cells comprise immune presenting cells.

[0067] In one embodiment, the immune cell is selected from the group consisting of dendritic cells (DC), T lymphocytes, macrophages and natural killer (NK) cells, and any combination thereof.

[0068] In one embodiment, the immune cells comprise bone marrow-derived dendritic cells (BMDCs).

[0069] In one embodiment, the immune cells comprise CD8-positive cells and / or CD4-positive cells.

[0070] In one embodiment, the method of the present application can be in vitro or ex vivo. In one embodiment, the method of the present application can be for non-preventive and non-therapeutic purposes.

[0071] On the other hand, the present application also provides a method for activating immune cells, which comprises administering the pharmaceutical combination, vaccine and / or kit of the present application.

[0072] In one embodiment, the immune cells comprise immune presenting cells.

[0073] In one embodiment, the immune cell is selected from the group consisting of dendritic cells (DC), T lymphocytes, macrophages and natural killer (NK) cells, and any combination thereof.

[0074] In one embodiment, the immune cells comprise bone marrow-derived dendritic cells (BMDCs).

[0075] In one embodiment, the immune cells comprise CD8-positive cells and / or CD4-positive cells.

[0076] In one embodiment, the immune cells comprise immune cells in lymph nodes and / or spleen.

[0077] In one embodiment, the effect of the activation of the immune cells by administering the drug combination comprises an effect selected from the following groups, compared to the immune cells to which the drug combination is not administered: increasing the expression level of the antigen recognition receptor of the immune cells, increasing the expression level of the nuclear factor κB (NF-κB) protein of the immune cells, increasing the expression and / or secretion level of the cytokines of the immune cells and increasing the proportion of mature immune cells, and any combination thereof.

[0078] In one embodiment, the antigen recognition receptor comprises a pattern recognition receptor (PRR).

[0079] In one embodiment, the antigen recognition receptor comprises a Toll-like receptor (TLR).

[0080] In one embodiment, the antigen recognition receptor comprises TLR1, TLR2 and / or TLR6.

[0081] In one embodiment, the expression of TLR4 in the immune cells administered with the drug combination is substantially unchanged compared to immune cells not administered with the drug combination.

[0082] In one embodiment, the cytokine comprises a pro-inflammatory cytokine.

[0083] In one embodiment, the cytokines comprise interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β, and / or interferon (IFN)-γ.

[0084] In one embodiment, the mature immune cells comprise CD80-positive cells, CD86-positive cells and / or effector memory cells.

[0085] In one embodiment, the ratio of mature immune cells comprises the ratio of CD80-positive and / or CD86-positive immune cells to CD11c-positive immune cells.

[0086] In one embodiment, the proportion of mature immune cells comprises the proportion of immune cells with high expression of CD44 and low expression of CD62L to immune cells that are CD8 positive.

[0087] In one embodiment, the method of the present application can be in vitro or ex vivo. In one embodiment, the method of the present application can be for non-preventive and non-therapeutic purposes.

[0088] On the other hand, the present application also provides the use of the drug combination of the present application, the vaccine of the present application and / or the kit of the present application in the preparation of a drug, wherein the drug is used to enhance innate immunity and / or specific immune response.

[0089] In one embodiment, administration of the drug combination promotes dendritic cell maturation and / or increases cytokine secretion by lymphocytes compared to no administration of the drug combination.

[0090] In one embodiment, the use does not substantially induce a systemic inflammatory response.

[0091] In one embodiment, the use that does not substantially induce a systemic inflammatory response comprises that administration of the drug combination does not substantially increase the concentration of IFN-γ, TNF-α, macrophage chemoattractant protein-1 (MCP-1), IL-12p70, IL-10, IL-23, IL-27, IL-17A, IFN-β, granulocyte-macrophage colony-stimulating factor (GM-CSF) and / or IL-1α in the serum of the subject compared to not administering the drug combination.

[0092] In one embodiment, the use does not substantially induce hemolysis, heart damage, liver damage, spleen damage, lung damage, and / or kidney damage.

[0093] On the other hand, the present application also provides the pharmaceutical combination, vaccine and / or kit of the present application, which are used to enhance innate immunity and / or specific immune response.

[0094] In one embodiment, administration of the drug combination promotes dendritic cell maturation and / or increases cytokine secretion by lymphocytes compared to no administration of the drug combination.

[0095] In one embodiment, the use does not substantially induce a systemic inflammatory response.

[0096] In one embodiment, the use that does not substantially induce a systemic inflammatory response comprises that administration of the drug combination does not substantially increase the concentration of IFN-γ, TNF-α, macrophage chemoattractant protein-1 (MCP-1), IL-12p70, IL-10, IL-23, IL-27, IL-17A, IFN-β, granulocyte-macrophage colony-stimulating factor (GM-CSF) and / or IL-1α in the serum of the subject compared to not administering the drug combination.

[0097] In one embodiment, the use does not substantially induce hemolysis, heart damage, liver damage, spleen damage, lung damage, and / or kidney damage.

[0098] On the other hand, the present application also provides a method for enhancing innate immunity and / or specific immune response, comprising administering the pharmaceutical combination, vaccine and / or kit of the present application to a subject in need thereof.

[0099] In one embodiment, administration of the drug combination promotes dendritic cell maturation and / or increases cytokine secretion by lymphocytes compared to no administration of the drug combination.

[0100] In one embodiment, the method does not substantially induce a systemic inflammatory response.

[0101] In one embodiment, the method of not substantially inducing a systemic inflammatory response comprises administering the drug combination without substantially increasing the concentration of IFN-γ, TNF-α, macrophage chemoattractant protein-1 (MCP-1), IL-12p70, IL-10, IL-23, IL-27, IL-17A, IFN-β, granulocyte-macrophage colony stimulating factor (GM-CSF) and / or IL-1α in the serum of the subject, compared to not administering the drug combination.

[0102] In one embodiment, the method does not substantially induce hemolysis, heart damage, liver damage, spleen damage, lung damage, and / or kidney damage.

[0103] On the other hand, the present application also provides the use of the drug combination of the present application, the vaccine of the present application and / or the kit of the present application in the preparation of a drug, wherein the drug is used to prevent and / or treat tumors.

[0104] In one embodiment, the prevention and / or treatment of tumors comprises slowing down the rate of increase in tumor volume and / or reducing the volume of the tumor.

[0105] In one embodiment, said tumor comprises said tumor that was incompletely removed following tumor resection.

[0106] In one embodiment, inhibiting a tumor comprises re-occurrence of a tumor after clearance of said tumor.

[0107] In one embodiment, the tumor comprises a solid tumor.

[0108] In one embodiment, the tumor is selected from the group consisting of breast tumor, colon tumor, liver tumor, stomach tumor, kidney tumor, pancreatic tumor, ovarian tumor, lymphoma, osteosarcoma, glioma, prostate cancer and melanoma, and any combination thereof.

[0109] On the other hand, the present application also provides the drug combination, vaccine and / or kit of the present application, which are used to prevent and / or treat tumors.

[0110] In one embodiment, the prevention and / or treatment of tumors comprises slowing down the rate of increase in tumor volume and / or reducing the volume of the tumor.

[0111] In one embodiment, said tumor comprises said tumor that was incompletely removed following tumor resection.

[0112] In one embodiment, inhibiting a tumor comprises re-occurrence of a tumor after clearance of said tumor.

[0113] In one embodiment, the tumor comprises a solid tumor.

[0114] In one embodiment, the tumor is selected from the group consisting of breast tumor, colon tumor, liver tumor, stomach tumor, kidney tumor, pancreatic tumor, ovarian tumor, lymphoma, osteosarcoma, glioma, prostate cancer and melanoma, and any combination thereof.

[0115] On the other hand, the present application also provides a method for preventing and / or treating tumors, comprising administering the drug combination, vaccine and / or kit of the present application to a subject in need thereof.

[0116] In one embodiment, the prevention and / or treatment of tumors comprises slowing down the rate of increase in tumor volume and / or reducing the volume of the tumor.

[0117] In one embodiment, said tumor comprises said tumor that was incompletely removed following tumor resection.

[0118] In one embodiment, inhibiting a tumor comprises re-occurrence of a tumor after clearance of said tumor.

[0119] In one embodiment, the tumor comprises a solid tumor.

[0120] In one embodiment, the tumor is selected from the group consisting of breast tumor, colon tumor, liver tumor, stomach tumor, kidney tumor, pancreatic tumor, ovarian tumor, lymphoma, osteosarcoma, glioma, prostate cancer and melanoma, and any combination thereof.

[0121] On the other hand, the present application also provides a method for preparing the pharmaceutical combination, vaccine and / or kit of the present application, comprising providing the membrane derived from the bacterial inner membrane.

[0122] In one embodiment, the method further comprises providing the component derived from an organism other than the bacteria.

[0123] In one embodiment, the method further comprises mixing the membrane derived from the inner membrane of bacteria and the component derived from an organism other than the bacteria to provide an outer shell.

[0124] In one embodiment, a kernel is further provided.

[0125] In one aspect, the present invention provides a tumor vaccine for preventing postoperative cancer recurrence, wherein the tumor vaccine comprises a hybrid cell membrane shell and a core material, wherein the hybrid cell membrane shell comprises a Gram-negative bacterial inner membrane and a solid tumor cell membrane derived from surgical resection.

[0126] The tumor vaccine involved in the present invention is a nanoparticle with a core material wrapped in a hybrid membrane. The present invention uses the cell membrane of solid tumor tissue derived from surgical resection in the hybrid membrane as a tumor antigen, and creatively uses the inner membrane of Gram-negative bacteria in the hybrid membrane as an immune adjuvant. The two can be delivered together into the same dendritic cell, utilizing the uptake and presentation of tumor antigens, and can synergistically enhance the body's innate immunity and specific immunity. It also has a certain lymph node enrichment ability, has a significant effect in preventing recurrence after tumor resection surgery, prolongs the patient's survival, and provides a long-term protection mechanism.

[0127] The core material encapsulated in the tumor vaccine hybrid membrane involved in the present invention can help maintain the rigid structure and stability of the hybrid membrane, thereby maintaining the hybrid membrane's immune enhancement, lymphocyte enrichment, and prevention of tumor recurrence after resection surgery; at the same time, it can further encapsulate other bioactive components such as siRNA or chemotherapy drugs to achieve more functions.

[0128] In addition, the tumor vaccine involved in the present invention has good biological safety, readily available raw materials, and low preparation cost; it also has a wide range of applications and can be modified with various chemical groups on the vaccine surface according to actual needs, thus having broad application prospects.

[0129] In one embodiment, the molar ratio of the bacterial endomembrane to the protein in the solid tumor cell membrane derived from surgical resection is 1:100-100:1, for example, 1:100, 1:75, 1:50, 1:25, 1:10, 1:5, 1:3, 1:1, 1:0, 0:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1 or 100:1, etc., for example, it can be (2-3):1. Specific point values ​​within the above numerical range can be selected and will not be repeated here.

[0130] The molar ratio of the bacterial inner membrane to the protein in the solid tumor cell membrane derived from surgical resection is specifically selected to be in the numerical range of 1:100-100:1, wherein (2-3):1 is the numerical range with the best effect.

[0131] In one embodiment, the mass ratio of the hybrid cell membrane shell to the core material is 1:(1-10), for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, etc., for example, it can be 1:(4-6). Specific point values ​​within the above numerical range can be selected and will not be repeated here.

[0132] The mass ratio of the hybrid cell membrane shell to the core material is specifically selected to be in the numerical range of 1:(1-10), because within this range, the surface charge of the final product is closer to the surface charge of the hybrid cell membrane shell.

[0133] In one embodiment, the Gram-negative bacteria include any one or a combination of at least two of the genera Escherichia coli, Staphylococcus, Bacillus, Lactobacillus, Klebsiella, Brucella, Proteus, Acinetobacter or Pseudomonas.

[0134] The Gram-negative bacteria covered by this invention include, but are not limited to, the types listed above. The inner membranes of other Gram-negative bacteria can also achieve the technical effects described herein. The cell wall of Gram-negative bacteria is a multilayered structure, consisting, from the outside to the inside, of the outer membrane (OM) consisting of a phospholipid layer and a lipopolysaccharide layer; a thin peptidoglycan (PGN) layer in the middle; and the inner membrane (IM) (cytoplasmic membrane) consisting of a lipoprotein layer and a phospholipid layer. The inner membrane of Gram-negative bacteria is essentially free of endotoxins (lipopolysaccharides (LPS)), providing excellent biosafety.

[0135] In one embodiment, the tumor comprises any one or a combination of at least two of breast cancer, colon cancer, liver cancer, gastric cancer, kidney cancer, pancreatic cancer, ovarian cancer, lymphoma, osteosarcoma, glioma, prostate cancer or melanoma.

[0136] The tumors involved in the present invention include but are not limited to the above-mentioned types, and other types of tumors can be applied to the technical solutions involved in the present invention.

[0137] In one embodiment, the core material includes any one or a combination of at least two of PLGA, MOF, PCL, PAMAM, carbon nanotubes, graphene, gold nanoparticles, mesoporous silica nanoparticles, or iron oxide nanoparticles.

[0138] The polymers involved in the present invention include, but are not limited to, the types listed above. Other biocompatible polymers can also achieve the technical effects of the present invention. The hybrid cell membrane outer shell structure is a phospholipid bilayer, which can be easily wrapped around the surface of various core materials. The method is simple and can be combined according to actual needs.

[0139] In one embodiment, the particle size of the tumor vaccine for preventing postoperative cancer recurrence is 100-300 nm, for example, 100 nm, 150 nm, 200 nm, 250 nm or 300 nm, etc. Specific point values ​​within the above numerical range can be selected and will not be described in detail here.

[0140] In one embodiment, the core material of the tumor vaccine also encapsulates any one or a combination of at least two of immune checkpoint inhibitors, IDO inhibitors, siRNA or chemotherapy drugs; the combination of at least two, for example, a combination of immune checkpoint inhibitors and IDO inhibitors, a combination of siRNA and chemotherapy drugs, etc., and any other arbitrary combination methods can be selected, which will not be described here one by one.

[0141] In another aspect, the present invention provides a method for preparing the above-mentioned tumor vaccine for preventing postoperative cancer recurrence, characterized in that the preparation method comprises the following steps:

[0142] (1) Extract the inner membrane of Gram-negative bacteria and the cell membrane of solid tumors obtained by surgical resection;

[0143] (2) mixing the Gram-negative bacterial inner membrane extracted in step (1) with the solid tumor cell membrane obtained by surgical resection, and extruding the mixture using a liposome extruder to obtain hybrid membrane nanoparticles;

[0144] (3) The hybrid membrane microparticles obtained in step (2) are mixed with the nanoparticle cores and extruded using a liposome extruder to obtain the tumor vaccine for preventing postoperative cancer recurrence.

[0145] In one embodiment, the mixing temperature in step (2) is 20-45°C, for example, 20°C, 25°C, 30°C, 35°C, 40°C or 45°C, and the mixing time is 10-20min, for example, 10min, 12min, 15min, 18min or 20min, etc. Specific point values ​​within the above numerical range can be selected and will not be described one by one here.

[0146] In one embodiment, the pore size of the filter membrane of the liposome extruder in step (2) is 300-500 nm, for example, 300 nm, 350 nm, 400 nm, 450 nm or 500 nm, etc. Specific values ​​within the above numerical range can be selected and will not be described in detail here.

[0147] In one embodiment, the extrusion in step (2) refers to a cumulative back-and-forth extrusion of 10-15 times (one back-and-forth is counted as one time).

[0148] In one embodiment, the mixing temperature in step (3) is 20-45°C, for example, 20°C, 25°C, 30°C, 35°C, 40°C or 45°C, and the mixing time is 10-20min, for example, 10min, 12min, 15min, 18min or 20min, etc. Specific point values ​​within the above numerical range can be selected and will not be described one by one here.

[0149] In one embodiment, the pore size of the filter membrane of the liposome extruder in step (3) is 100-300 nm, for example, 100 nm, 150 nm, 200 nm, 250 nm or 300 nm, etc. Specific values ​​within the above numerical range can be selected and will not be described in detail here.

[0150] In one embodiment, the extrusion in step (3) refers to a cumulative back-and-forth extrusion of 10-15 times (one back-and-forth is counted as one time).

[0151] In one embodiment, the nanoparticle core of step (3) is prepared by a double emulsion method. Specifically, it can be exemplified as follows:

[0152] (1) Dissolve the core material in an organic solvent and then add sterile distilled water;

[0153] (2) ultrasonic emulsification of the mixture;

[0154] (3) then mixed with sodium cholate solution and ultrasonically emulsified;

[0155] (4) adding the emulsion dropwise to the sodium cholate solution and stirring;

[0156] (5) Rotary evaporation; centrifugation; washing with sterile distilled water and resuspending to obtain the nanoparticle core.

[0157] In one embodiment, the organic solvent includes any one of dichloromethane, chloroform, acetone or ethanol, or a combination of at least two thereof.

[0158] In one embodiment, the volume ratio of the organic solvent to sterile distilled water is (3-7):1, for example 3:1, 4:1, 5:1, 6:1 or 7:1, etc. Any specific point value within the above numerical range can be selected, and they will not be described one by one here.

[0159] In one embodiment, the temperature of the ultrasound in step (2) is 0-4°C, for example, 0°C, 1°C, 2°C, 3°C or 4°C, the time is 2-5min, for example, 2min, 3min, 4min or 5min, and the power is 20-30W, for example, 20W, 22W, 25W, 28W or 30W. Any specific point value within the above numerical range can be selected, and they will not be repeated here.

[0160] In one embodiment, the mass fraction of the sodium cholate solution in step (3) is 1-3%, such as 1%, 2% or 3%.

[0161] In one embodiment, the volume ratio of the sodium cholate solution to the organic solvent in step (3) is (1-3):1, such as 1:1, 2:1 or 3:1.

[0162] In one embodiment, the temperature of the ultrasound in step (3) is 0-4°C, for example, 0°C, 1°C, 2°C, 3°C or 4°C, the time is 4-6 min, for example, 4 min, 5 min or 6 min, and the power is 25-35 W, for example, 25 W, 28 W, 30 W, 32 W or 35 W. Any specific point value within the above numerical range can be selected, and they will not be repeated here.

[0163] In one embodiment, the mass fraction of the sodium cholate solution in step (4) is 0.3-0.7%, such as 0.3%, 0.5% or 0.7%.

[0164] In one embodiment, the volume ratio of the sodium cholate solution to the organic solvent in step (4) is (8-12):1, such as 8:1, 10:1 or 12:1.

[0165] In one embodiment, the stirring temperature in step (4) is 20-30°C, such as 20°C, 25°C or 30°C, and the time is greater than 10 minutes.

[0166] In another aspect, the present invention provides a use of the above-mentioned tumor vaccine for preventing postoperative cancer recurrence in the preparation of a drug for enhancing the body's innate immunity and specific immune response.

[0167] In another aspect, the present invention provides a use of the above-mentioned tumor vaccine for preventing postoperative cancer recurrence in the preparation of a medicament for promoting dendritic cells to secrete pro-inflammatory cytokines.

[0168] In another aspect, the present invention provides a use of the above-mentioned tumor vaccine for preventing postoperative cancer recurrence in the preparation of a drug for promoting dendritic cell maturation.

[0169] In another aspect, the present invention provides a use of the above-mentioned tumor vaccine for preventing postoperative cancer recurrence in the preparation of a medicament for promoting IFN-γ secretion by splenic lymphocytes.

[0170] In another aspect, the present invention provides a use of the above-mentioned tumor vaccine for preventing postoperative cancer recurrence in the preparation of a drug for stimulating the systemic immune system to secrete inflammatory factors and chemokines.

[0171] Compared with the prior art, the present invention has the following beneficial effects:

[0172] (1) The tumor vaccine involved in the present invention is a hybrid membrane-encapsulated polymer nanoparticle. The present invention uses the cell membrane of solid tumor tissue derived from surgical resection in the hybrid membrane as a tumor antigen, and creatively uses the inner membrane of Gram-negative bacteria in the hybrid membrane as an immune adjuvant. The two can be co-delivered to the same dendritic cell, utilizing the uptake and presentation of tumor antigens, which can enhance the body's innate immunity and specific immunity. It also has a certain lymph node enrichment ability, has a significant effect in preventing tumor recurrence after resection surgery, prolongs the patient's survival, and provides a long-term protective mechanism. In animal experiments, it effectively inhibited postoperative tumor recurrence in tumor-bearing mice undergoing tumor resection for breast cancer, with an inhibition rate of up to 83.3%; it completely inhibited postoperative tumor recurrence in tumor-bearing mice undergoing tumor resection for colon cancer, with an inhibition rate of up to 100%.

[0173] (2) The core material encapsulated in the tumor vaccine hybrid membrane of the present invention can help maintain the rigid structure and stability of the hybrid membrane, thereby maintaining the hybrid membrane's efficacy in immune enhancement, lymphocyte enrichment, and prevention of tumor recurrence after resection surgery; at the same time, it can further encapsulate other bioactive components such as siRNA or chemotherapy drugs to achieve more functions.

[0174] (3) In addition, the tumor vaccine involved in the present invention has good biological safety, the raw materials for preparation are easily available, and the preparation cost is low. In order to obtain a drug combination or vaccine with good safety, the present application uses the components of the bacterial inner membrane as one of the components of the drug combination; and it has a wide range of applications, and various chemical groups can be modified on the vaccine surface according to actual needs, and it has broad application prospects.

[0175] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0176] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0177] Figure 1 Shown are transmission electron microscopy images of EM-NPs, TM-NPs, and HM-NPs hybrid membrane nanoparticles, with a scale bar of 100 nm;

[0178] Figure 2 Shown are the dynamic light scattering results of HM-NPs hybrid membrane nanoparticles;

[0179] Figure 3 A-3C shows the comparison of IL-6, TNF-α, and IL-1β levels in the supernatant of the control group, EM-NPs group, TM-NPs group, Mix NPs group, and HM-NPs group (A is the IL-6 level, B is the TNF-α level, and C is the IL-1β level);

[0180] Figure 4 Shown are comparisons of CD80 and CD86 levels in the supernatants of the control group, EM-NPs group, TM-NPs group, Mix NPs group, and HM-NPs group (left is CD80 level, right is CD86 level);

[0181] Figure 5 The graph shows the results of ELISPOT kit detection of IFN-γ secretion in the supernatant of the control group, EM-NPs group, TM-NPs group, Mix NPs group and HM-NPs group;

[0182] Figure 6 Shown are the results of statistical analysis of the number of spots in the control group, EM-NPs group, TM-NPs group, Mix NPs group, and HM-NPs group using CTL-ImmunoSpot Plate Reader software;

[0183] Figure 7 A-7M shows the results of the secretion levels of inflammatory factors and chemokines in the serum of mice in the control group, EM-NPs group, TM-NPs group, Mix NPs group, and HM-NPs group (A, B, C, D, E, F, G, H, I, J, K, L, and M correspond to IL-6, IFN-γ, TNF-α, MCP-1, IL-12p70, IL-1β, IL-10, IL-23, IL-27, IL-17A, IFN-β, GM-CSF, and IL-1α, respectively);

[0184] Figure 8 Shown is an in vivo imaging diagram of the lymph node enrichment effect of the product prepared in Example 1;

[0185] Figure 9 Shown is a statistical result diagram of the lymph node enrichment effect of the product prepared in Example 1 in vivo;

[0186] Figure 10 Shown are the results of hemolysis test of HM-NPs hybrid membrane nanoparticles;

[0187] Figure 11Shown is a flow chart of the experimental operation of Example 6;

[0188] Figure 12 Shown are the in vivo imaging images of tumor fluorescence intensity in tumor-bearing mice in the control group, EM-NPs group, TM-NPs group, Mix NPs group, and HM-NPs group;

[0189] Figure 13 Shown are the changes in tumor volume over time in each tumor-bearing mouse in the control group, EM-NPs group, TM-NPs group, Mix NPs group, and HM-NPs group;

[0190] Figure 14 Shown are the changes in tumor volume over time in tumor-bearing mice in the control group, EM-NPs group, TM-NPs group, Mix NPs group, and HM-NPs group;

[0191] Figure 15 Shown are the survival curves of tumor-bearing mice in the control group, EM-NPs group, TM-NPs group, Mix NPs group, and HM-NPs group;

[0192] Figure 16 Shown is a flow chart of the experimental operation of Example 7;

[0193] Figure 17 Shown are photos of tumor changes over time in tumor-bearing mice in the control group, Mix NPs group, and HM-NPs group;

[0194] Figure 18 Shown are the survival curves of tumor-bearing mice in the control group, Mix NPs group, and HM-NPs group;

[0195] Figure 19 Shown are the results of the changes in tumor size over time in each group of mice during the postoperative re-challenge test;

[0196] Figure 20 A-20D shows the comparison of IL-6, TNF-α, IL-1β, and IFN-γ expression levels in the serum of mice in each group during the postoperative rechallenge test (A, B, C, and D correspond to IL-6, TNF-α, IL-1β, and IFN-γ, respectively);

[0197] Figure 21 A-21C shows a comparison of the levels of IL-6, TNF-α, and IL-1β in the supernatant of products with different membrane protein concentration ratios in hybrid membrane nanoparticles (A, B, and C correspond to IL-6, TNF-α, and IL-1β, respectively).

[0198] Figure 22Shown is a Venn diagram illustrating the different protein expression profiles of (left) EM and (right) TM from four batches. The sum of the numbers in each large circle represents the total number of proteins expressed in four biologically independent replicates (n=4). A total of 2,302 proteins were detected in the EM samples and 5,353 proteins were detected in the TM samples. The overlapping areas of the circles represent co-expressed proteins. E1, E2, E3, and E4 represent four batches of EM samples, respectively. T1, T2, T3, and T4 represent four batches of TM samples, respectively.

[0199] Figures 23A-23C Shown are graphs of proinflammatory cytokine concentrations in BMDC supernatants after 24 hours of incubation with the indicated EM to TM membrane protein mass ratios (EM:TM) at 1:100, 1:75, 1:50, 1:25, 1:10, 1:5, 1:3, 1:1, 1:0, 0:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1, or 100:1. (A to C) IL-6, TNF-α, and IL-1β production were analyzed using corresponding specific ELISA kits. n = 3 biologically independent replicates. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01. ns indicates no significant difference.

[0200] Figure 24 A-24C shows the physicochemical properties of three membrane vesicles before encapsulation with PLGA nanoparticle cores. (A) TEM images of EM, TM, and HM vesicles. Scale bar, 100 nm. Experiments were performed at least three times (N = 3). (B) Hydrated particle size and (C) Zeta potential of EM, TM, and HM vesicles. n = 3, biologically independent replicates. Data are expressed as mean ± SD.

[0201] Figure 25 Shown are the LPS contents of EM-NPs, TM-NPs, and HM-NPs. Most LPS is removed during the EM extraction process. There were no significant differences in LPS content between the three membrane NPs (p > 0.9999) (n = 3, biologically independent replicates). Data are presented as mean ± SD.

[0202] Figure 26 A-26B shows a schematic diagram of a core / shell HM-NP. (A) Representative TEM image of a single HM-NP. (B) HM-NP core size (diameter) and shell thickness based on TEM statistics (n = 26 nanoparticles). The HM-NP core diameter is 86.83 ± 7.41 nm (inner line), and the shell thickness is 14.16 ± 2.07 nm (outer line). Experiments were performed at least three times (N = 3). Scale bar, 20 nm. Data are expressed as mean ± SD.

[0203] Figure 27 A-27B shows the physicochemical stability of HM-NPs before and after lyophilization and resuspension in PBS. (A and B) Size and zeta potential of HM-NPs before and after lyophilization and resuspension in PBS in 10% wt% sucrose (n = 3, biologically independent replicates). Data are expressed as mean ± SD.

[0204] Figure 28 Figure 28B shows the particle size and charge of EM-NPs, TM-NPs, and HM-NPs stored in PBS at 4°C for different time intervals. (A) Size and (B) zeta potential of EM-NPs, TM-NPs, and HM-NPs stored in PBS for 3 weeks (n = 3, biologically independent replicates). Data are presented as mean ± SD.

[0205] Figures 29A-29E Shown are schematic diagrams of the construction of cancer vaccine HM-NPs and characterization results of EM-NPs, TM-NPs, and HM-NPs. (A) Preparation of HM-NPs. Tumors were surgically removed from tumor-bearing mice to obtain TMs. Expanded Escherichia coli DH5α was treated with lysozyme to remove the cell wall, and protoplasts were obtained by low-speed centrifugation. EMs were then prepared using extraction buffer. PLGA NPs were prepared using a double emulsion method. The two membranes were mixed and repeatedly physically extruded through a liposome extruder with a 400 nm pore size filter. PLGA polymer nanoparticle cores were then added and repeatedly physically extruded through a liposome extruder with a 200 nm pore size filter to produce HM-NPs. (B) TEM images of EM-NPs, TM-NPs, and HM-NPs. Scale bar, 100 nm. (C) Size distribution and zeta potential of EM-NPs, TM-NPs, and HM-NPs. (D) Western blot analysis of characteristic proteins in EM-NPs, TM-NPs, and HM-NPs. Each lane was loaded with equal amounts of protein (10 μg, n=3 biologically independent samples). (E) Schematic diagram of the cross-linked chromatin matrix with FtsZ (dark arrows, 5 nm gold NPs) and Na + / K + TEM images of EM-NPs, TM-NPs, and HM-NPs immunogold-labeled with ATPase (light arrows, 10 nm gold NPs) stained with uranyl acetate. Scale bar, 100 nm. Experiments were performed at least three times (N=3).

[0206] Figure 30Shown are the full protein expression profiles of EM-NPs, TM-NPs, and HM-NPs. Protein profiles of EM-NPs, TM-NPs, and HM-NPs were resolved by SDS-PAGE followed by Coomassie blue staining. TM rectangles: Representative proteins on the tumor cell plasma membrane (TM) from resected autologous tumor tissue; EM rectangles: Protein bands within a prominent molecular weight range, derived from the Escherichia coli cell plasma membrane (EM). Equal amounts of protein (10 μg) were loaded in each lane. Experiments were performed three times with three biological replicates per group.

[0207] Figures 31A-31B Shown are TEM images of HM-NPs with FtsZ (dark arrows, 5 nm gold NPs) and Na + / K + Immunogold labeling of α-ATPase (light arrow, 10 nm gold NPs) followed by negative staining with uranyl acetate. (A) The percentage of hybrid membrane nanoparticles labeled with both 5 nm and 10 nm gold nanoparticles (AuNPs) in the HM-NP formulation was calculated using three TEM observation fields. A total of 20 hybrid membrane nanoparticles were counted as shown in (B). The experiment was performed at least three times. Scale bar, 100 nm. Data are expressed as mean ± SD.

[0208] Figure 32 Shown are confocal fluorescence images of EM-NPs-RhoB, TM-NPs-RhoB, and HM-NPs-RhoB internalized into the lysosomal compartment of BMDCs. BMDCs were incubated with each of the three fluorescently labeled membrane nanoparticles for 1 hour and observed by confocal microscopy. The experiment was performed three times, with three biological replicates per group. Rhodamine B-loaded NPs: second column; lysosomal compartment: first column; bright field (BF): third column; merged image: fourth column. Scale bar, 20 μm.

[0209] Figure 33 Shown are the results of a Pearson correlation coefficient analysis between the lysosomal compartment and membrane NPs. n = 20 BMDCs were included, and statistical significance was calculated by one-way analysis of variance with Bonferroni's multiple comparison test. Data are expressed as mean ± SD. The experiment was performed three times, with three biological replicates per group.

[0210] Figure 34Shown are the results of BMDCs co-incubated with bacterial endometrium (EM) at various concentrations (0, 0.12, 0.25, 0.50, and 1.00 mg / mL). Cell supernatants were collected at the indicated time points (0, 3, 8, 24, and 48 hours) and the levels of the proinflammatory cytokine IL-6 in the BMDCs were measured using a specific ELISA kit. In all groups, IL-6 secretion reached a plateau after 24 hours of co-incubation. The curve represents the change in IL-6 secretion over 48 hours. IL-6 production was positively correlated with the concentration of EM-NPs. Data are expressed as mean ± standard deviation. The experiment was performed three times, with three biological replicates per group.

[0211] Figures 35A-35K Shown are images of HM-NPs enhancing tumor antigen uptake, activating TLR expression on the surface of BMDCs, and co-delivering antigen and adjuvant to BMDCs. (A) Colocalization analysis of EM-NPs, TM-NPs, and HM-NPs (Rhodamine B; second column) with lysosomal compartments (first column) in BMDCs by confocal microscopy (1-hour incubation). Third column, bright field. Scale bar, 5 μm. (B) Cellular uptake of Rhodamine B-loaded membrane NPs after 8 hours of incubation with BMDCs, as assessed by flow cytometry (n = 6, biologically independent samples). (C) Western blot analysis of a range of BMDC membrane TLR and NF-κB proteins in membrane-coated nanoparticles. Equal amounts of protein were loaded in each lane. (D) Schematic diagram of activation of TLRs and their downstream signaling pathways. (E) Concentrations of three proinflammatory cytokines in BMDC supernatants after 24 hours of incubation with membrane NPs (n = 6, biologically independent samples). (H and I) After incubation with membrane NPs for 24 h, CD80 + or CD86 + Flow cytometric analysis of BMDCs (n = 6, biologically independent samples). (J) Immunofluorescence experiments of confocal laser scanning microscopy images of mouse BMDCs treated with mixed NPs or HM-NPs for 8 hours. DAPI-labeled nuclei: first column; Na + / K + -ATPase-labeled TM: second column; FtsZ-labeled EM: third column. Scale bar, 20 μm. (K) Pearson correlation coefficient analysis between TM and EM in each sample (n = 20 cells). Experiments were performed in triplicate. Data are expressed as mean ± standard deviation. Statistical significance was calculated by one-way ANOVA with Bonferroni's multiple comparison test; ***p < 0.001, ****p < 0.0001, and ns indicates no significant difference.

[0212] Figures 36A-36BShown are validation results demonstrating enhanced accumulation of HM-NPs in draining lymph nodes (LNs). (A) Ex vivo optical imaging of inguinal LNs. IR-780-labeled EM-NPs, TM-NPs, or HM-NPs were subcutaneously injected into Balb / c mice. LNs (control group, n = 5 mice; EM-NPs, TM-NPs, or HM-NPs-treated groups, n = 6 mice each) were harvested 12 hours later. (B) Quantification of mean fluorescence intensity in panel A. Data are expressed as mean ± standard deviation. Experiments were performed at least three times. Statistical significance was calculated by two-way ANOVA with Bonferroni's multiple comparison test; ****p < 0.0001, ns indicates no significant difference.

[0213] Figures 37A-37G The figure shows the validation results of HM-NPs promoting DC maturation and splenic T cell activation in LN in vivo. (A and B) CD80 in inguinal lymph nodes of 4T1 tumor-bearing mice after subcutaneous inoculation with membrane NPs + and CD86 + Flow cytometric analysis of DCs. The proportion of autologous membrane antigen-specific T cells was determined in IFN-γ ELISPOT assays and flow cytometry. (CD) Representative results of IFN-γ ELISPOT assays and the corresponding quantitative number of spots in 4T1 tumor-bearing mice (5 mice per group). (EF) CD3 in the spleen of 4T1 tumor-bearing mice after subcutaneous inoculation with membrane NPs. + CD8 + IFNγ + Percentage of cells (5 mice per group). (G) HM-NPs induced a low-level systemic inflammatory response. Inflammatory cytokines and chemokines in serum were detected using Luminex magnetic bead-based ELISA kits in the ProcartaPlex multiplex immunoassay (n = 5 mice per group). The experiment was performed at least three times. Data are expressed as mean ± SD. In Figures AD, statistical significance was calculated by one-way analysis of variance using Bonferroni's multiple comparison test. In G, the heat map of the Luminex-based ProcartaPlex multiplex immunoassay was grouped and analyzed by two-way analysis of variance and Bonferroni's multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference.

[0214] Figure 38Figure A-38I shows the validation results of HM-NPs eliciting a low-level systemic inflammatory response. The vaccine formulation was subcutaneously injected into mice on days 3, 5, and 9 after surgery (n = 5 mice per group). Serum concentrations of inflammatory cytokines and chemokines were measured using Luminex magnetic bead-based ELISA kits and analyzed using a BD Accuri C6 FACS flow cytometer and ProcartaPlex Analysis software. Data are expressed as mean ± standard deviation. Experiments were performed in triplicate. Statistical significance was calculated by one-way ANOVA with Bonferroni's multiple comparison test.

[0215] Figures 39A-39E Figures show the validation results of HM-NPs inducing tumor regression in a 4T1-Luc mouse tumor model. (A) Scheme showing the animal experiment design. Female BALB / c mice were subcutaneously inoculated with 4T1-Luc mammary tumor cells. When the tumor volume reached approximately 300 mm 3 , they were surgically removed. The mice were then immunized with the designated vaccine formulations on days 3, 5, and 9 after surgery, and the tumor growth of the mice was monitored weekly by the IVIS imaging system. (B) In vivo bioluminescence images of mice bearing 4T1-Luc tumors on different days. (C and D) Single and average tumor growth curves. (Complete response, CR) refers to the disappearance of all target lesions, the absence of new lesions, and normal tumor markers, which last for at least 4 weeks. (E) Survival curves of mice treated with each vaccine (n = 12 mice per group). The experiment was performed at least three times. The data are expressed as mean ± standard deviation. The statistical significance of the experiment was calculated by two-way analysis of variance (ANOVA), Bonferroni's multiple comparison test for growth curves, and the log-rank test (Mantel-Cox) for comparison of survival curves, ***p < 0.001, ****p < 0.0001.

[0216] Figures 40A-40G Figures show the validation results of HM-NP vaccination in suppressing tumor recurrence in various mouse tumor models. (A) Scheme showing the design of the animal experiment. Mice were subcutaneously inoculated with tumor cells. When the tumor volume reached approximately 300 mm 3When the tumor was resected, it was surgically removed. Mice were immunized with different vaccine formulations on days 3, 5, and 9 after surgery and observed for 60 days (B). Images of mice in the control, mixed NP, and HM-NP groups. Average tumor growth curve (C), single tumor growth curve (D), and survival curve (E) of each group in the CT26 tumor model (n = 12 mice per group). CR, complete remission. (F) Survival curve of each group in the B16F10 melanoma model (n = 10 mice per group). (G) Survival curve of each group in the EMT-6 tumor model (n = 10 mice per group). Data are expressed as mean ± standard deviation. The experiment was performed at least three times. Statistical significance was analyzed by two-way analysis of variance (ANOVA), Bonferroni's multiple comparison test for growth curves, and log-rank test (Mantel-Cox) for survival curves, ***p < 0.001, ****p < 0.0001.

[0217] Figures 41A-41B The figure shows the results of the validation of the therapeutic efficacy of HM-NP compared with the three formulations of MPLA and tumor membrane preparation or HM vesicle preparation in the mouse CT-26 tumor model. Female BALB / c mice were subcutaneously inoculated with CT-26 colon adenocarcinoma cells (2×10 per mouse). 5 The three MPLA vaccine formulations included TM-NPs + free MPLA, MPLA anchored to the tumor membrane (TM-MPLA-NPs), and PLGA nanoparticles containing MPLA with tumor membranes (TM-NPs@MPLA). In addition, hybrid membrane vesicles (HM vesicles) without PLGA were subcutaneously inoculated into postoperative mice to compare the therapeutic potential of hybrid membrane-coated PLGA NPs (HM NPs) in preventing tumor recurrence. (A) Tumor growth curves of individual mice in each group and (B) average tumor growth curves and survival curves. CR: complete remission. Data are expressed as mean ± standard deviation. Experiments were performed at least three times. Statistical significance was calculated by two-way analysis of variance (ANOVA), Bonferroni's multiple comparison test for growth curves, and log-rank (Mantel-Cox) test for logarithmic survival curves. *p < 0.05, **p < 0.01, ****p < 0.0001, ns indicates no significant difference.

[0218] Figures 42A-42IFigure 1: HM-NP vaccination provides long-term protective antitumor immunity; both innate and adaptive immunity are crucial for vaccine efficacy. (A) Postoperative mice were immunized with HM-NPs. On day 60, mice treated with HM-NPs were randomly divided into different groups and inoculated with saline, CT-26, or 4T1 cells (n = 12 mice per group). Individual tumor growth curves of mice treated with HM-NPs and re-inoculated with CT-26 or 4T1 cells 60 days later (B). ELISA was used to measure the levels of proinflammatory cytokines (C) IL-6, (D) IL-1β, (E) TNF-α, and (F) IFN-γ in the serum of mice inoculated with CT-26 or 4T1 cells (n = 12 mice per group). Splenocytes were isolated from naive mice or HM-NP-treated mice with tumor eradication (90 days after primary tumor inoculation). (GH) Representative scatter plots and CD8 + Percentage (%) of T cell subsets with high expression of CD44 and low expression of CD62L in T cells (n = 12 mice per group). The experiment was performed at least three times. (I) Depletion of specific immune cell subsets (NK cells, macrophages, CD8 + T cells or CD4 + The mean tumor growth curves of mice expressing HM-NPs (T cells) were analyzed to explore their relative contribution to the observed anti-tumor efficacy of HM-NPs (n = 5 mice per group). Data are presented as mean ± standard deviation. Statistical significance was analyzed using a two-way ANOVA with Bonferroni's multiple comparison test, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0219] Figure 43 The results of depletion of specific immune cell subsets during HM-NP treatment are shown in Figure 3. As shown in Table 3, the corresponding immune cell subsets were knocked down by intraperitoneal injection of depletion antibodies starting one day before the start of HM-NP treatment. The peripheral blood of mice was analyzed by flow cytometry to confirm the expression of macrophages (CSF1R antibody), NK cells (ASGM1 antibody), CD8 + T cells (CD8 antibodies) or CD4 + Depletion of T cells (CD4 antibody) (n=5, biologically independent samples).

[0220] Figures 44A-44B The figure shows the validation results that both innate and adaptive immunity are required for effective tumor suppression after HM-NP treatment. Female BALB / c mice were subcutaneously inoculated with CT-26 colon adenocarcinoma cells (2×10 per mouse). 5 When the tumor volume reaches about 300 mm 3The mice were evaluated on day 2 after surgery and randomly divided into six groups (n=5 mice per group). In addition to the control group, mice were immunized on days 3, 5, and 9 after surgery. Depletion antibodies against designated surface markers were injected intraperitoneally one day before starting HM-NP treatment to deplete mouse macrophages (CSF1R antibody), NK cells (ASGM1 antibody), CD8 + T cells (CD8 antibodies) or CD4 + T cells (CD4 antibody). (A) Survival curves and (B) individual tumor growth curves. CR, complete remission (n = 5 mice per group). Statistical significance was calculated using two-way analysis of variance (ANOVA) and Bonferroni's multiple comparison test for growth curves. Log-rank (Mantel-Cox) test for comparison of survival curves: *p < 0.05, **p < 0.01, ns indicates no significant difference.

[0221] Figure 45 Shown are graphs showing the results of a hemolysis assay at various HM-NP concentrations. A range of HM-NP concentrations were added to erythrocytes. Hemolysis was assessed and calculated after 3 hours. Erythrocytes treated with PBS (0% hemolysis) and three times with water (100% hemolysis) served as controls. No hemolysis was observed at any of the HM-NP concentrations tested (n = 3, biologically independent replicates).

[0222] Figure 46 Shown are representative microscopic images of H&E-stained sections of major organs (i.e., heart, liver, spleen, lung, and kidney) from each group of mice at the endpoint of tumor regression in the 4T1-Luc model. The experiment was performed three times, with three biological replicates per group. Scale bar, 50 μm.

[0223] Figure 47 Figures A-47D show the results of evaluating liver and kidney function in all groups of mice at the end of the tumor regression experiment in the 4T1-Luc model. (A to D) Biochemical analysis of serum from mice at the end of the tumor regression experiment in the 4T1-Luc model (n = 6 mice per group). ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CREA, creatinine. Data are expressed as mean ± standard deviation. Statistical significance was analyzed by one-way analysis of variance using Bonferroni's multiple comparison test.

[0224] Figure 48 Shown is the distribution of E. coli inner membrane proteins.

[0225] Figures 49A-49BFigures show the results of a hybrid membrane nanovaccine, prepared from the inner membrane of bacteria from the same genus, inhibiting postoperative recurrence in a colon cancer model. (A) Morphology of hybrid membrane nanoparticles prepared from different bacterial inner membranes and tumor membranes. (B) Survival curves of mice following immunotherapy with hybrid membrane nanoparticles prepared from different bacteria.

[0226] Figures 50A-50C Shown are the results of HM-NP hybrid membrane nanovaccines based on different tumor cell membranes inhibiting postoperative recurrence in corresponding tumor models. (A) Schematic diagram of tumor model construction and immunization procedure. (B) Morphology of hybrid membrane nanoparticles prepared from different tumor cell membranes and bacterial endomembranes. (C) Survival curves of mice following immunotherapy with HM-NP hybrid membrane nanovaccines prepared from liver cancer, gastric cancer, kidney cancer, pancreatic cancer, ovarian cancer, lymphoma, osteosarcoma, glioma, prostate cancer, and melanoma cell membranes.

[0227] Figure 51 Shown is a hybrid membrane wrapped with PLGA polymer, extruded through a liposome extruder with a pore size of 200 nm. The surface potential of the hybrid membrane shell can be adjusted from +50 mV to -50 mV by doping with polymers with different charges. DETAILED DESCRIPTION

[0228] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0229] Definition of terms

[0230] In this application, the term "bacterial inner membrane" generally refers to a membrane structure of bacteria. For example, bacteria can have multiple structures, from the outside to the inside, including the cell wall, bacterial outer membrane, bacterial inner membrane, and cytoplasm. For example, the bacterial inner membrane in this application can only be the inner bacterial membrane structure near the cytoplasm.

[0231] In this application, the term "pharmaceutical combination" generally refers to a product produced by mixing or combining one or more active ingredients. The term "pharmaceutical combination" can be administered to a patient separately, together, or sequentially (without specific time limits), wherein such administration provides therapeutically effective levels of the first membrane component of the present application and the component derived from an organism other than the bacteria in the patient's body.

[0232] In this application, the term "membrane component" generally refers to a component having a membrane structure. For example, the membrane component of this application may include natural or synthetic biological membrane components. For example, the membrane component of this application may not include phospholipid components, and the membrane component of this application may only include membrane proteins, membrane lipids, membrane sugar molecules, membrane glycoproteins, or membrane lipoproteins of biological membranes. For example, the membrane component of this application may include functionally active fragments of membrane components as well as other arbitrary structural domains.

[0233] In this application, the term "organism" generally refers to an organism having a boundary structure. For example, the organism in this application can be a living object. For example, the organism in this application can be a cellular organism with a cell structure or a viral organism without a cell structure. For example, the organism in this application can include bacteria, prokaryotes, eukaryotes, as well as the tissues, cells, or non-cellular structures mentioned above.

[0234] In this application, the term "immunogenicity" generally refers to the ability to elicit an immune response (stimulate the production of specific antibodies and / or the proliferation of specific T cells).

[0235] In this application, the term "biocompatibility" generally refers to a material having compatibility with a host. For example, biocompatibility may refer to a material not eliciting an abnormal blood reaction, immune reaction, and / or tissue reaction.

[0236] In this application, the terms "Gram-positive bacteria" or "Gram-negative bacteria" generally refer to the fact that bacteria can be divided into two main categories based on the basic characteristics of the Gram stain reaction: G-positive (G+) and G-negative (G-). After staining, the bacterial cells of the former can still retain the blue-purple color of the initial crystal violet stain, while after staining, the bacterial cells of the latter lose the initial crystal violet color and take on a complex red or sandy red.

[0237] In this application, the term "interleukin-6" or "IL-6" generally refers to a type of cytokine. For example, IL-6 can be found in GenBank Accession No. P05231. The IL-6 protein herein may also encompass functionally active fragments thereof, and is not limited to substances comprising functionally active fragments of IL-6 produced after processing and / or modification in cells. For example, the IL-6 herein may include functionally active fragments of IL-6 as well as any other domains.

[0238] In this application, the term "interferon-γ" or "IFN-γ" generally refers to a type of cytokine. For example, IFN-γ can be found in GenBank Accession No. P01579. The IFN-γ protein of this application may also include functionally active fragments thereof, and is not limited to substances containing functionally active fragments of IFN-γ produced after processing and / or modification in cells. For example, the IFN-γ of this application may include functionally active fragments of IFN-γ as well as any other domains.

[0239] In this application, the term "interleukin-1β" ​​or "IL-1β" generally refers to a type of cytokine. For example, IL-1β can be found in GenBank accession number P01584. The IL-1β protein of this application can also include functionally active fragments thereof, and is not limited to substances containing functionally active fragments of IL-1β produced after processing and / or modification in cells. For example, the IL-1β of this application can include functionally active fragments of IL-1β as well as any other domains.

[0240] In this application, the term "tumor necrosis factor-α" or "TNF-α" generally refers to a type of cytokine. For example, TNF-α can be found in GenBank Accession No. P01375. The TNF-α protein herein may also encompass functionally active fragments thereof, and is not limited to substances comprising functionally active fragments of TNF-α produced after processing and / or modification in cells. For example, the TNF-α herein may include functionally active fragments of TNF-α as well as any other domains.

[0241] In this application, the term "lipopolysaccharide" generally refers to Lipopolysaccharide (abbreviated as LPS in English). For example, LPS can refer to a component of the outer cell wall of Gram-negative bacteria, which is a substance composed of lipids and polysaccharides (glycolipids).

[0242] In this application, the term "FtsZ" generally refers to a protein in the bacterial inner membrane. For example, FtsZ can be used to detect whether a hybrid membrane contains membrane components derived from the bacterial inner membrane. For example, FtsZ can be found in GenBank accession number P0A9A6. For example, the FtsZ of the present application can include functionally active fragments of FtsZ and other arbitrary domains. The bacterial inner membrane can also include lipopolysaccharide (LPS, commonly known as endotoxin) and phosphatidylethanolamine (PE). The intracellular membrane can also include secondary lipid classes, such as N-acylated PE (acyl-PE) and O-acylated PG (acyl-PG), etc., and can be used to detect whether a hybrid membrane contains membrane components derived from the bacterial inner membrane. The lipopolysaccharide molecule is located in the outer layer of the cell outer membrane and can be composed of two parts: a hydrophilic polysaccharide chain and a hydrophobic lipid A. The amphiphilic molecule phosphatidylethanolamine is the most abundant lipid molecule in the inner membrane of Gram-negative bacteria, accounting for approximately 77%; the anionic lipid phosphatidylglycerol (PG) accounts for approximately 13%, and cardiolipin (CL) accounts for approximately 10%. The bacterial inner membrane is rich in lipoproteins and lipids. Phosphatidylethanolamine is arguably the most important phospholipid molecule in Gram-negative bacteria, playing a crucial role in maintaining normal cell morphology, cell growth and differentiation, and substance synthesis. Bacterial membranes can contain a diverse array of amphiphilic lipids, including the common phospholipids phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin, as well as less common phosphatidylcholine, phosphatidylinositol, and a variety of other membrane lipids, such as ornithine lipids, glycolipids, and sphingolipids.

[0243] In this application, the term "Na+ / K+-ATPase" generally refers to a protein of a cell membrane. For example, it can be a protein of a mammalian cell membrane. For example, Na+ / K+-ATPase can be used to detect whether a hybrid membrane contains membrane components from sources other than the bacterial inner membrane. For example, Na+ / K+-ATPase can be found in GenBank Accession No. P13637. For example, the Na+ / K+-ATPase of this application can include a functionally active fragment of the Na+ / K+-ATPase and any other domains. Detailed Description of the Invention

[0245] On the one hand, the present application provides a drug combination, which may include a first membrane component, which may include a membrane derived from the inner membrane of bacteria, and the drug combination may also include components derived from other organisms other than the bacteria. For example, the drug combination of the present application may include a membrane derived from the inner membrane of bacteria, and immunogenic substances derived from other organisms. For example, the drug combination of the present application may include a membrane derived from the inner membrane of bacteria, and a tumor antigen. For example, for the convenience of preparation, the drug combination of the present application may include a membrane derived from the inner membrane of bacteria, and a shell of a cell membrane and / or non-cellular membrane structure from which the antigen is derived.

[0246] For example, the other organisms described herein may comprise cells. For example, the other organisms described herein may comprise non-cellular structures.

[0247] For example, other organisms of the present application may comprise mammalian cells. For example, other organisms of the present application may comprise non-mammalian cells, such as prokaryotic cells.

[0248] For example, the other organisms of the present application may comprise tumor cells. For example, the other organisms of the present application may comprise solid tumor cells.

[0249] For example, the other organisms of the present application can be selected from the following group: breast cancer cells, colon cancer cells, liver cancer cells, gastric cancer cells, kidney cancer cells, pancreatic cancer cells, ovarian cancer cells, lymphoma cells, osteosarcoma cells, glioma cells, prostate cancer cells and melanoma cells, and any combination thereof.

[0250] For example, the component of the other organism may comprise an immunogenic component. For example, the component of the other organism may be capable of eliciting an immune response to the organism.

[0251] For example, the component of the other organism may comprise a component derived from a cell membrane of the organism.

[0252] For example, the component of the other organism may comprise a tumor antigen or a functionally active fragment thereof. For example, the component of the other organism may comprise an immunogenic protein or an immunogenic fragment thereof on the cell membrane of the other organism.

[0253] For example, the component of the other organism may comprise a protein or a functionally active fragment thereof selected from the following group: antigen peptide transporter 1, H-2 class II histocompatibility antigen gamma chain, tyrosine protein kinase SYK, high affinity immunoglobulin epsilon receptor subunit gamma, Ras-related C3 botulinum toxin substrate 2, tyrosine protein kinase BTK, receptor type tyrosine protein phosphatase C, Na + / K +-ATPase, ATP5A (IM CVa), ubiquinone cytochrome c reductase core protein I (IM Core I), VDAC1 / porin (OM Porin), matrix cyclophilin D (Matrix CypD) and intermembrane space cytochrome c (IMS Cytc), and any combination thereof.

[0254] For example, the pharmaceutical composition of the present application may include a second membrane fraction comprising a membrane derived from a cell membrane of the other organism. For example, the second membrane fraction of the present application may be a cell membrane comprising an immunogenic protein. For example, the second membrane fraction of the present application may also be prepared by combining an immunogenic protein with a natural and / or artificial cell membrane.

[0255] For example, in the pharmaceutical combination of the present application, the bacteria may include Gram-negative bacteria and / or Gram-positive bacteria.

[0256] For example, the drug regimen of the present application, the antibacterial is selected from the genus of following group: Escherichia coli, Staphylococcus, Bacillus, Lactobacillus, Klebsiella, Brucella, Proteus, Acinetobacter and Pseudomonas, and above-mentioned in any combination.For example, the drug regimen of the present application, the antibacterial is selected from the genus of following group: Escherichia coli (Escherichia), Staphylococcus (Staphylococcus), Bacillus (Bacillus), Lactobacillus (Lactobacillus) and Pseudomonas (Pseudomonas).For example, the drug regimen of the present application, the antibacterial can comprise Escherichia coli and / or Staphylococcus aureus.

[0257] For example, in the drug combination of the present application, the inner membrane of the bacteria may comprise a protein or a functionally active fragment thereof selected from the following group: FtsZ, YhcB, YidC, NlpI, MsbA, TatA, MlaA, TolQ and YebE, and any combination thereof.

[0258] For example, the drug combination of the present application may further comprise a core. For example, the core of the drug combination of the present application may be used to support the membrane component of the present application.

[0259] For example, the core of the pharmaceutical combination of the present application may also include a biocompatible material. For example, the core of the pharmaceutical combination of the present application may also include a synthetic material. For example, materials with granular and / or non-granular structures that can be covered or partially covered by the membrane of the present application, as known in the art, may be used in the core of the pharmaceutical combination of the present application.

[0260] For example, the core of the drug combination of the present application can also include a substance selected from the following groups: polylactic acid-co-glycolic acid (PLGA), metal-organic framework materials (MOF), polycaprolactone (PCL), polyamide-amine (PAMAM), carbon nanotubes, graphene, gold nanoparticles, mesoporous silica nanoparticles, iron oxide nanoparticles, silver nanoparticles, tungsten nanoparticles, manganese nanoparticles, platinum nanoparticles, quantum dots, aluminum oxide nanoparticles, hydroxyapatite nanoparticles, lipid nanoparticles (LNP), DNA nanostructures, nanohydrogels, rare earth fluoride nanocrystals and NaYF4 nanoparticles, and any combination thereof.

[0261] For example, the core of the drug combination of the present application may further comprise a substance selected from the following group: an immune adjuvant, an immune checkpoint inhibitor, a nucleic acid molecule and a chemotherapeutic agent, and any combination thereof.

[0262] For example, the core of the drug combination of the present application may further comprise monophosphoryl lipid A (MPLA), R848, CpG, poly(I:C), and any combination thereof.

[0263] For example, the core of the drug combination of the present application may also include doxorubicin, paclitaxel, docetaxel, gemcitabine, capecitabine, cyclophosphamide, fluorouracil, pemetrexed, raltitrexed, bleomycin, daunorubicin, doxorubicin, vincristine and etoposide, as well as any combination thereof.

[0264] For example, the core of the drug combination of the present application may further comprise an indoleamine 2,3-dioxygenase (IDO) inhibitor.

[0265] For example, the core of the drug combination of the present application may also contain small interfering RNA (siRNA).

[0266] For example, the diameter of the kernel of the drug regimen of the present application can be approximately 60 to approximately 100 nanometers.For example, the size of the kernel of the drug regimen of the present application can not affect the effect of the drug regimen of the present application basically.For example, the diameter of the kernel of the drug regimen of the present application can be approximately 60 to approximately 100 nanometers, approximately 70 to approximately 100 nanometers, approximately 80 to approximately 100 nanometers, approximately 90 to approximately 100 nanometers, approximately 60 to approximately 90 nanometers, approximately 70 to approximately 90 nanometers or approximately 80 to approximately 90 nanometers.For example, the diameter of the kernel of the drug regimen of the present application can be approximately 86 nanometers.For example, the diameter of the kernel of the application can be measured by transmission electron microscopy.

[0267] For example, the pharmaceutical combination of the present application may comprise a shell, and the shell may comprise the first membrane component and the second membrane component.

[0268] For example, the shell of the present application may include a membrane formed by the fusion of the first membrane component and the second membrane component. For example, the shell of the present application may include natural and / or artificially synthesized biofilms, as well as proteins of bacterial inner membranes and proteins of biofilms of other organisms. For example, the shell of the present application may include natural or artificially synthesized biofilms, as well as immunogenic proteins of bacterial inner membranes and immunogenic proteins of other organisms. For example, for ease of preparation, the shell of the present application may be composed of the following groups: bacterial inner membranes containing immunogenic proteins and other biofilms containing immunogenic proteins. For example, components derived from bacterial inner membranes may be used to enhance the immune response of a subject; for example, components derived from other organisms may be used to induce an immune response of a subject to the other organisms. For example, using tumor cell membranes as components derived from other organisms may be used to induce and / or enhance the immune response of a subject to the tumor.

[0269] For example, the thickness of the shell of the present application can be approximately 10 to approximately 20 nanometers.For example, the thickness of the shell of the present application can be approximately 10 to approximately 20 nanometers, approximately 11 to approximately 20 nanometers, approximately 12 to approximately 20 nanometers, approximately 13 to approximately 20 nanometers, approximately 14 to approximately 20 nanometers, approximately 15 to approximately 20 nanometers, approximately 16 to approximately 20 nanometers, approximately 17 to approximately 20 nanometers, approximately 18 to approximately 20 nanometers, approximately 19 to approximately 20 nanometers, approximately 10 to approximately 15 nanometers, approximately 11 to approximately 15 nanometers, approximately 12 to approximately 15 nanometers, approximately 13 to approximately 15 nanometers or approximately 14 to approximately 15 nanometers.For example, the thickness of the shell of the present application can be approximately 14 nanometers.

[0270] For example, the diameter of the shell of the present application can be about 100 nanometers.For example, the thickness or diameter of the shell of the present application can be measured by transmission electron microscopy.

[0271] For example, the surface potential (Zeta potential) of the shell of the present application may be about +50 mV to about -50 mV.

[0272] For example, the surface potential (Zeta potential) of the shell of the present application can be about -21 mV. For example, the surface potential (Zeta potential) of the shell can be about +50 mV to about -50 mV, about -15 to about -50 mV, about -21 to about -50 mV, about -25 to about -50 mV, about +50 mV to about -25 mV, about +50 mV to about -21 mV, about -15 to about -25 mV, about -17 to about -25 mV, about -19 to about -25 mV, about -21 to about -25 mV, about -23 to about -25 mV, about -15 to about -21 mV, about -17 to about -21 mV, or about -19 to about -21 mV.

[0273] For example, the mass ratio of the first membrane component to the second membrane component of the housing of the present application may be 1:100 to 100:1.

[0274] For example, the mass ratio of the first membrane component to the second membrane component of the shell of the present application can be 1:100-100:1, for example, 1:100, 1:75, 1:50, 1:25, 1:10, 1:5, 1:3, 1:1, 1:0, 0:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1 or 100:1. For example, the mass ratio of the first membrane component to the second membrane component of the shell of the present application can be 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 0:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1 or 100:1.

[0275] For example, the presence and / or ratio of the first membrane fraction and the second membrane fraction in the pharmaceutical combination of the present application can be confirmed by Western blotting and / or immunogold staining.

[0276] For example, the pharmaceutical composition of the present application may comprise particles, and the particles may comprise the core and the shell.

[0277] For example, the mass ratio of the shell to the core material of the pharmaceutical combination of the present application can be about 1:1 to about 1:10.

[0278] For example, the mass ratio of the shell to the core material of the pharmaceutical combination of the present application can be about 1:4 to about 1:6. For example, the mass ratio of the shell to the core material of the pharmaceutical combination of the present application can be about 1:1 to about 1:10, about 1:2 to about 1:10, about 1:4 to about 1:10, about 1:6 to about 1:10, about 1:8 to about 1:10, about 1:1 to about 1:6, about 1:2 to about 1:6, or about 1:4 to about 1:6. For example, the mass ratio of the shell to the core material of the pharmaceutical combination of the present application can be about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 or about 1:10.

[0279] For example, the lipopolysaccharide (LPS) content of the pharmaceutical combination of the present application may not be significantly different from the lipopolysaccharide content of mammalian cells. For example, the pharmaceutical combination of the present application may substantially not contain lipopolysaccharide from bacteria.

[0280] For example, the particles of the drug combination of the present application may have a diameter of about 70 to about 120 nanometers.

[0281] For example, the diameter of the particle of the drug regimen of the present application can be about 100 nanometers.For example, the diameter of the particle of the drug regimen of the present application can be about 70 to about 120 nanometers, about 80 to about 120 nanometers, about 90 to about 120 nanometers, about 100 to about 120 nanometers, about 110 to about 120 nanometers, about 70 to about 100 nanometers, about 80 to about 100 nanometers or about 90 to about 100 nanometers.For example, the diameter can be measured by transmission electron microscope (TEM).

[0282] For example, the hydrated particle size of the particles can be from about 150 nm to about 250 nm. For example, the hydrated particle size of the particles can be from about 180 nm. For example, the hydrated particle size of the particles can be from about 150 nm to about 250 nm, from 160 nm to about 250 nm, from 180 nm to about 250 nm, from 200 nm to about 250 nm, from 220 nm to about 250 nm, from 240 nm to about 250 nm, from about 150 nm to about 180 nm, or from 160 nm to about 180 nm.

[0283] For example, the surface potential (Zeta potential) of the particle of the present application can be about +50mV to about -50mV. For example, the surface potential (Zeta potential) of the particle of the present application can be about -21mV. For example, the surface potential (Zeta potential) of the particle of the present application can be about +50mV to about -50mV, about -15 to about -50mV, about -21 to about -50mV, about -25 to about -50mV, about +50mV to about -25mV, about +50mV to about -21mV, about -15 to about -25mV, about -17 to about -25mV, about -19 to about -25mV, about -21 to about -25mV, about -23 to about -25mV, about -15 to about -21mV, about -17 to about -21mV, or about -19 to about -21mV.

[0284] For example, the hydrated particle size and / or surface potential of the particles can be measured by a dynamic light scattering (DLS) instrument.

[0285] For example, the particles can be able to maintain stability in solution. For example, the stability of the particles can include that the hydrated particle size and / or surface potential of the particles after being stored for a period of time are not significantly different from those before the period of time.

[0286] For example, the stability of the particles may include that the hydrated particle size and / or surface potential of the particles after being stored in phosphate buffered saline (PBS) at 4 degrees Celsius for a period of time are not significantly different from those before the period of time.

[0287] For example, the stability of the particles can include that the hydrated particle size and / or surface potential of the particles after being stored in phosphate buffered saline (PBS) at 4 degrees Celsius for a period of time are not significantly different from those before the period of time. For example, the period of time can be no less than about 21 days. For example, the period of time can be no less than about 21 days, no less than about 20 days, no less than about 15 days, no less than about 10 days, no less than about 5 days, no less than about 4 days, no less than about 3 days, no less than about 2 days, or no less than about 1 day.

[0288] For example, the drug combination of the present application may further comprise a substance selected from the following groups: an immune adjuvant, an immune checkpoint inhibitor, a nucleic acid molecule, a chemotherapeutic agent, and a photosensitizer, and any combination thereof. For example, the above components in the drug combination of the present application may be administered simultaneously and / or separately.

[0289] For example, the drug combination of the present application may further comprise monophosphoryl lipid A (MPLA). For example, the drug combination of the present application may further comprise an indoleamine 2,3-dioxygenase (IDO) inhibitor. For example, the drug combination of the present application may further comprise small interfering RNA (siRNA).

[0290] On the other hand, the present application provides a vaccine that may comprise the drug combination of the present application.

[0291] On the other hand, the present application provides a kit that may contain the drug combination of the present application and / or the vaccine of the present application.

[0292] On the other hand, the present application also provides a method for preparing the pharmaceutical combination, vaccine and / or kit of the present application, which may comprise providing the membrane derived from the bacterial inner membrane.

[0293] For example, the method of the present application may further comprise providing the components derived from organisms other than the bacteria.

[0294] For example, the method of the present application may further comprise mixing the membrane derived from the inner membrane of bacteria and the components derived from organisms other than the bacteria to provide an outer shell.

[0295] For example, the method of the present application may also include providing a kernel.

[0296] In another aspect, the present application provides a method for enhancing the uptake of a target antigen by immune cells, which may include providing a drug combination, wherein the drug combination may include a first membrane component, wherein the first membrane component may include a membrane derived from the inner membrane of a bacterium, and the drug combination may further include the target antigen. For example, the method of the present application may be in vitro or ex vivo. For example, the method of the present application may be for non-preventive, non-therapeutic, and / or non-diagnostic purposes.

[0297] For example, in the method of the present application, the drug combination may comprise the drug combination of the present application.

[0298] For example, in the method of the present application, the immune cells may include immune presenting cells.

[0299] For example, in the method of the present application, the immune cells may include the following groups: dendritic cells (DC), T lymphocytes, macrophages and natural killer (NK) cells, and any combination thereof.

[0300] For example, in the method of the present application, the immune cells may include bone marrow-derived dendritic cells (BMDCs).

[0301] For example, in the method of the present application, the immune cells may include CD8-positive cells and / or CD4-positive cells.

[0302] In another aspect, the present application provides a method for activating immune cells, which may comprise administering the pharmaceutical combination, the vaccine, and / or the kit described herein. For example, the method may be in vitro or ex vivo. For example, the method may be for non-preventive, non-therapeutic, and / or non-diagnostic purposes.

[0303] For example, in the method of the present application, the immune cells may include immune presenting cells.

[0304] For example, in the method of the present application, the immune cells may include the following groups: dendritic cells (DC), T lymphocytes, macrophages and natural killer (NK) cells, and any combination thereof.

[0305] For example, in the method of the present application, the immune cells may include bone marrow-derived dendritic cells (BMDCs).

[0306] For example, in the method of the present application, the immune cells may include CD8-positive cells and / or CD4-positive cells.

[0307] For example, in the method of the present application, the immune cells may include the immune cells in the lymph nodes and / or spleen.

[0308] For example, in the method of the present application, the effect of the activation of the immune cells administered with the drug combination can be selected from the following groups: increasing the expression level of the antigen recognition receptor of the immune cells, increasing the expression level of the nuclear factor kappa B (NF-κB) protein of the immune cells, increasing the expression and / or secretion level of the cytokines of the immune cells and increasing the proportion of mature immune cells, and any combination thereof. For example, the increase comprises an increase of about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% in the effect of the immune cells administered with the drug combination compared to the immune cells to which the drug combination is not administered. For example, the increase comprises an increase in the expression level of an antigen recognition receptor of the immune cell administered with the drug combination by about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% compared to the immune cell not administered with the drug combination. For example, the increase comprises an increase in the expression level of nuclear factor kappa B (NF-κB) protein of the immune cell administered with the drug combination by about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% compared to the immune cell not administered with the drug combination. For example, the increase comprises an increase in the expression and / or secretion level of the cytokines of the immune cells of the immune cells administered with the drug combination compared to the immune cells not administered with the drug combination by about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%. For example, the increase comprises an increase in the proportion of mature immune cells of the immune cells administered with the drug combination compared to the immune cells not administered with the drug combination by about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.

[0309] For example, in the method of the present application, the antigen recognition receptor may comprise a pattern recognition receptor (PRR).

[0310] For example, in the method of the present application, the antigen recognition receptor may comprise a Toll-like receptor (TLR).

[0311] For example, in the method of the present application, the antigen recognition receptor may comprise TLR1, TLR2 and / or TLR6.

[0312] For example, in the method of the present application, the expression of TLR4 in the immune cells administered with the drug combination may remain substantially unchanged compared to the immune cells not administered with the drug combination.

[0313] For example, in the method of the present application, the cytokine may comprise a pro-inflammatory cytokine.

[0314] For example, in the method of the present application, the cytokines may include interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β and / or interferon (IFN)-γ.

[0315] For example, in the method of the present application, the mature immune cells may include CD80-positive cells, CD86-positive cells and / or effector memory cells.

[0316] For example, in the method of the present application, the ratio of mature immune cells may include the ratio of CD80-positive and / or CD86-positive immune cells to CD11c-positive immune cells.

[0317] For example, in the method of the present application, the ratio of mature immune cells may include the ratio of immune cells with high expression of CD44 and low expression of CD62L to immune cells that are CD8 positive.

[0318] On the other hand, the present application provides a method for enhancing innate immunity and / or specific immune response, which may comprise administering the pharmaceutical combination, vaccine and / or kit of the present application to a subject in need thereof.

[0319] For example, in the method of the present application, administration of the drug combination can promote dendritic cell maturation and / or increase cytokine secretion by lymphocytes compared to not administering the drug combination.

[0320] For example, in the method of the present application, the application may not substantially induce a systemic inflammatory response.

[0321] For example, in the method of the present application, the application of not substantially inducing a systemic inflammatory response may comprise administering the drug combination without substantially increasing the concentration of IFN-γ, TNF-α, macrophage chemoattractant protein-1 (MCP-1), IL-12p70, IL-10, IL-23, IL-27, IL-17A, IFN-β, granulocyte-macrophage colony-stimulating factor (GM-CSF) and / or IL-1α in the serum of the subject, compared to not administering the drug combination.

[0322] For example, in the method of the present application, the application may substantially not induce hemolytic reaction, heart damage, liver damage, spleen damage, lung damage and / or kidney damage.

[0323] On the other hand, the present application provides the pharmaceutical combination, vaccine and / or kit of the present application, which can be used to enhance innate immunity and / or specific immune response.

[0324] For example, in the application of the present application, compared with not administering the drug combination, administering the drug combination can promote dendritic cell maturation and / or increase cytokine secretion by lymphocytes.

[0325] For example, in the application of the present application, the application may not substantially induce a systemic inflammatory response.

[0326] For example, in the application of the present application, the application that does not substantially induce a systemic inflammatory response may include that the administration of the drug combination does not substantially increase the concentration of IFN-γ, TNF-α, macrophage chemoattractant protein-1 (MCP-1), IL-12p70, IL-10, IL-23, IL-27, IL-17A, IFN-β, granulocyte-macrophage colony-stimulating factor (GM-CSF) and / or IL-1α in the serum of the subject compared to when the drug combination is not administered.

[0327] For example, in the application of the present application, the application may substantially not induce hemolytic reaction, heart damage, liver damage, spleen damage, lung damage and / or kidney damage.

[0328] On the other hand, the present application provides the use of the drug combination of the present application, the vaccine of the present application and / or the kit of the present application in the preparation of a drug, which can be used to enhance innate immunity and / or specific immune response.

[0329] For example, in the application of the present application, compared with not administering the drug combination, administering the drug combination can promote dendritic cell maturation and / or increase cytokine secretion by lymphocytes.

[0330] For example, in the application of the present application, the application may not substantially induce a systemic inflammatory response.

[0331] For example, in the application of the present application, the application that does not substantially induce a systemic inflammatory response may include that the administration of the drug combination does not substantially increase the concentration of IFN-γ, TNF-α, macrophage chemoattractant protein-1 (MCP-1), IL-12p70, IL-10, IL-23, IL-27, IL-17A, IFN-β, granulocyte-macrophage colony-stimulating factor (GM-CSF) and / or IL-1α in the serum of the subject compared to when the drug combination is not administered.

[0332] For example, in the application of the present application, the application may substantially not induce hemolytic reaction, heart damage, liver damage, spleen damage, lung damage and / or kidney damage.

[0333] On the other hand, the present application also provides a method for preventing and / or treating tumors, which may comprise administering the drug combination, vaccine and / or kit of the present application to a subject in need thereof.

[0334] For example, in the method of the present application, the prevention and / or treatment of tumors may include slowing down the rate of increase in tumor volume and / or reducing the volume of the tumor.

[0335] For example, in the method of the present application, the tumor may include the tumor that was not completely removed after tumor resection. For example, after a patient's tumor is removed by surgery, the tumor cells and / or tumor tissue that were not completely removed may develop into a tumor again, and this tumor may also be prevented and / or treated by the method of the present application. For example, in the method of the present application, the tumor may include a tumor that is regenerated after the tumor is cleared. For example, after a patient's tumor is removed by surgery, even if the tumor is completely removed, the patient may regenerate a tumor, such as if the patient is prone to tumors, and this newly regenerated tumor may also be prevented and / or treated by the method of the present application. For example, this newly regenerated tumor has at least one identical antigen as the original tumor.

[0336] For example, in the method of the present application, the tumor may comprise a solid tumor.

[0337] For example, in the method of the present application, the tumor can be selected from the following group: breast tumor, colon tumor, liver tumor, stomach tumor, kidney tumor, pancreatic tumor, ovarian tumor, lymphoma, osteosarcoma, glioma, prostate cancer and melanoma, and any combination thereof.

[0338] On the other hand, the present application also provides the drug combination, vaccine and / or kit of the present application, which can be used to prevent and / or treat tumors.

[0339] For example, in the application of the present application, the prevention and / or treatment of tumors may include slowing down the rate of increase in tumor volume and / or reducing the volume of the tumor.

[0340] For example, in the application of the present application, the tumor may include the tumor that was not completely removed after tumor resection.

[0341] For example, in the application of the present application, suppressing a tumor may include the recurrence of a tumor after the tumor is eliminated.

[0342] For example, in the application of the present application, the tumor may comprise a solid tumor.

[0343] For example, in the application of the present application, the tumor can be selected from the following group: breast tumor, colon tumor, liver tumor, stomach tumor, kidney tumor, pancreatic tumor, ovarian tumor, lymphoma, osteosarcoma, glioma, prostate cancer and melanoma, and any combination thereof.

[0344] On the other hand, the present application also provides the use of the drug combination of the present application, the vaccine of the present application and / or the kit of the present application in the preparation of drugs, which can be used to prevent and / or treat tumors.

[0345] For example, in the application of the present application, the prevention and / or treatment of tumors may include slowing down the rate of increase in tumor volume and / or reducing the volume of the tumor.

[0346] For example, in the application of the present application, the tumor may include the tumor that was not completely removed after tumor resection.

[0347] For example, in the application of the present application, suppressing a tumor may include the recurrence of a tumor after the tumor is eliminated.

[0348] For example, in the application of the present application, the tumor may comprise a solid tumor.

[0349] For example, in the application of the present application, the tumor can be selected from the following group: breast tumor, colon tumor, liver tumor, stomach tumor, kidney tumor, pancreatic tumor, ovarian tumor, lymphoma, osteosarcoma, glioma, prostate cancer and melanoma, and any combination thereof.

[0350] Without intending to be bound by any theory, the following examples are merely intended to illustrate the products, preparation methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.

[0351] Example

[0352] The Elisa kits involved in the following examples were purchased from Abcam, USA; CD80 and CD86 antibodies were purchased from BioLegend, USA; ELISPOT kits were purchased from Beijing Dakoway Biotechnology Co., Ltd.; and ProcartaPlexmultiplex immunoassay kits were purchased from Thermo Fisher Scientific, USA.

[0353] The experimental animals used in the following examples were female BALB / c mice, 6-8 weeks old, weighing 16-18 g, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and housed in a pathogen-free animal room at the National Center for Nanoscience and Technology. All animal experiments were conducted in strict accordance with the guidelines approved by the National Institutional Animal Care and Use Committee (IACUC).

[0354] The following examples use the inner membrane of Escherichia coli (E. coli DH5α) as a research model for the inner membrane of Gram-negative bacteria, the cell membrane of cells in 4T1-luciferase triple-negative breast cancer tissue derived from surgical resection or the cell membrane of cells in CT26 colon cancer tissue as research models for the cell membrane of solid tumors derived from surgical resection, and PLGA as a research model for the core material.

[0355] The drug combination of this application uses mixed Escherichia coli plasma membranes (EM) and autologous tumor membranes (TM) from resected tumor tissue to generate hybrid membrane nanoparticle vaccines (HM-NPs) for antigen and adjuvant delivery to antigen presenting cells (APCs). This personalized tumor vaccine is tailored to safely enhance the innate immune response and maximize anti-tumor effects while avoiding side effects. In the current study, this application demonstrated that the presence of EM can significantly increase DC uptake (p < 0.0001) of tumor antigens, activate Toll-like receptors (TLRs) on their plasma membranes and promote their maturation in vitro. This application confirmed the upregulation of stimulatory markers of DCs in lymph nodes (LNs) and the subsequent activation of tumor membrane antigen-specific T cells in vivo. In the postoperative recurrence prevention model, HM-NPs achieved significant anti-tumor immune effects in 4T1-Luc, B16F10, EMT-6 and CT-26 tumor models. In addition, in the CT26 colon tumor model, HM-NPs can induce a strong tumor-specific immune response, which not only prolongs the survival of animals after surgery, but also has a long-term protective effect against tumor re-stimulation (up to 3 months). The HM-NPs of this application are an effective autologous tumor vaccine that can successfully activate innate and adaptive immune responses, thereby significantly inhibiting tumor recurrence with negligible side effects. The current research shows that hybrid membrane vaccine technology may be transformed into an effective clinical treatment strategy for postoperative cancer treatment.

[0356] Example 1

[0357] In this example, four membrane nanoparticle systems were prepared, namely: ① nanoparticles formed by encapsulating PLGA with the inner membrane of Escherichia coli (hereinafter referred to as EM-NPs); ② nanoparticles formed by encapsulating PLGA with the cell membrane of cells in 4T1-luciferase triple-negative breast cancer tissue (hereinafter referred to as TM-NPs); ③ nanoparticles obtained by mixing the products of ① and ② (hereinafter referred to as Mix NPs); and ④ nanoparticles formed by encapsulating PLGA with a hybrid membrane composed of the inner membrane of Escherichia coli and tumor cell membrane (hereinafter referred to as HM-NPs). The total membrane protein concentration equivalents of the four products were consistent, and the concentration equivalents of the two membrane proteins in Mix NPs and HM-NPs were each consistent.

[0358] The preparation method of HM-NPs is as follows:

[0359] (1) Extracting the inner membrane of E. coli EM, the operation is as follows: E. coli is inoculated into LB liquid medium and cultured overnight to an OD600 value of 1.2; lysozyme with a final concentration of 2 mg / mL is added and incubated with E. coli at 37°C for 1 hour to lyse the cell wall; centrifugation is performed at 3000g for 5 minutes at 4 degrees Celsius, and the supernatant is discarded to obtain protoplasts; extraction buffer is added, and the inner membrane of E. coli is obtained by density gradient centrifugation; after resuspending, the mixture is stored at -80°C for later use;

[0360] (2) Extracting cell membrane TM from 4T1-luciferase triple-negative breast cancer tissues: 200,000 tumor cells were subcutaneously inoculated into each BALB / c mouse, and the cells were kept in the same place until the tumor volume reached 300 mm. 3 The tumor tissue was surgically removed under sterile conditions. The obtained tumor tissue was initially minced with scissors, and then a solution containing collagenase IV (1.0 mg mL -1 ), DNase (0.1 mg mL -1 ) and hyaluronidase (0.1 mg mL -1 ) GBSS solution at 37°C for 15 minutes to digest the tumor tissue; the digested tissue was ground on a 70 μm filter, then filtered through a 70 μm filter, and the filtrate was centrifuged and resuspended to obtain a single-cell suspension; an extraction buffer containing mannitol, sucrose, bovine serum albumin (BSA), Tris-HCl, EGTA, and a phosphatase-protease inhibitor cocktail was added, and the cells were then ultrasonically disrupted using a cell disruptor at 30W for 5 minutes under ice conditions, and the cells were centrifuged at 3000g, 10,000g, and 100,000g, respectively, to finally obtain tumor tissue-derived cell membranes; the cell membranes were resuspended and frozen at -80°C until use;

[0361] (3) 3 mg of extracted E. coli inner membrane EM and 1 mg of extracted surgically removed tumor cell membrane TM were mixed in PBS and shaken at 37°C for 15 min. The mixed membrane solution was then passed through a liposome extruder with a filter membrane pore size of 400 nm for a total of 13 times (one back and forth counted as one time) to obtain HM hybrid membrane microparticles;

[0362] (4) Dissolve 10 mg of PLGA in 1 mL of dichloromethane, and then add 0.2 mL of sterile distilled water; the mixture is ultrasonically emulsified on ice at 25 W for 3 min; then mix with 2 mL of 1% sodium cholate solution (surfactant) and ultrasonically emulsify on ice at 30 W for 5 min; add the emulsion dropwise to 10 mL of 0.5% sodium cholate solution and stir at room temperature for 15 min; use a rotary evaporator to evaporate at 37°C for 10 min; centrifuge the emulsion at 11,000 g for 15 min at room temperature; wash twice with sterile distilled water and resuspend to obtain a PLGA polymer nanoparticle solution;

[0363] (5) 50 μL of the 2 mg / mL HM hybrid membrane microparticles prepared in step (3) was mixed with 500 μL of the 1 mg / mL PLGA polymer nanoparticle solution prepared in step (4) and passed through a liposome extruder with a filter membrane pore size of 200 nm for a total of 13 times to obtain HM-NPs hybrid membrane nanoparticles.

[0364] The preparation method of EM-NPs is as follows:

[0365] (1) 4 mg of the prepared bacterial inner membrane EM solution was placed in a shaker at 37°C for 10 min; then, the solution was passed through a liposome extruder with a filter membrane of 400 nm in pore size, and extruded back and forth 13 times (one back and forth counted as one time);

[0366] (2) Referring to step (4) of the preparation method of HM-NPs, PLGA nanoparticles were prepared using the double emulsion method. 50 μL of the prepared 2 mg / mL bacterial endomembrane EM was mixed with 500 μL of the prepared 1 mg / mL PLGA polymer nanoparticle solution. The mixture was then passed through a liposome extruder with a filter membrane pore size of 200 nm, and extruded back and forth 13 times (one back and forth counted as one time) to finally obtain EM-NPs.

[0367] The preparation method of TM-NPs is as follows:

[0368] (1) 4 mg of the prepared surgically removed tumor cell membrane TM solution was placed on a shaker at 37°C for 10 min; then, the solution was passed through a liposome extruder with a filter membrane of 400 nm in pore size, and squeezed back and forth 13 times (one back and forth counted as one time);

[0369] (2) Referring to step (4) of the preparation method of HM-NPs, PLGA nanoparticles were prepared using the double emulsion method. 50 μL of the prepared 2 mg / mL surgically removed tumor cell membrane TM was mixed with 500 μL of the prepared 1 mg / mL PLGA polymer nanoparticle solution. The mixture was then passed through a liposome extruder with a filter membrane pore size of 200 nm and extruded back and forth 13 times (one back and forth counted as one time) to finally obtain TM-NPs.

[0370] The preparation method of Mix NPs is as follows:

[0371] (1) The prepared EM-NPs and TM-NPs were mixed at 37°C, where the concentrations of the two membrane proteins were kept consistent with those in HM-NPs.

[0372] The prepared EM-NPs, TM-NPs and HM-NPs hybrid membrane nanoparticles were observed using a transmission electron microscope (TECNAI G2 20S-TWIN, 200KV, FEI, USA). Figure 1 As shown, the results show that the three types of membrane nanoparticles are all regular round in shape and relatively uniform in size. The scale bar in the figure is 100 nm.

[0373] The prepared HM-NPs hybrid membrane nanoparticles were characterized by dynamic light scattering (Zetasizer NanoZS). Figure 2 As shown in the figure, it can be seen that the particle size of the prepared HM-NPs hybrid membrane nanoparticles is mainly distributed between 100-300nm, and reaches a peak at 164.2nm (accounting for 15.8%), indicating that the prepared hybrid membrane nanoparticles have a good particle size distribution, which is conducive to entering cells to exert biological effects.

[0374] Example 2

[0375] This example explores the effects of the four products prepared in Example 1 on enhancing innate immunity and specific immunity, and the specific contents include:

[0376] 1. Experiment on promoting BMDC to secrete pro-inflammatory cytokines:

[0377] (1) Extraction of bone marrow-derived dendritic cells (BMDCs) from BALB / c mice;

[0378] (2) BMDCs were cultured to day 5, resuspended in 1640 complete medium, and 40,000 cells were seeded per well in a 96-well plate. EM-NPs, TM-NPs, Mix NPs, and HM-NPs solutions with a final concentration of 1 mg / mL were then added for co-incubation. Three parallel wells were set up in each group. An equal volume of 1640 complete medium was also added as a control group (Control, Ctrl);

[0379] (3) After incubation at 37°C for 24 h, the supernatant of each culture medium was removed;

[0380] (4) Elisa kit was used to detect the levels of IL-6, TNF-α and IL-1β in the supernatant.

[0381] The results are as follows Figure 3As shown in A-3C, it can be seen that HM-NPs hybrid membrane nanoparticles have the best potential to enhance the body's innate immunity compared with other groups.

[0382] 2. Experiments to promote BMDC maturation:

[0383] (1) Extraction of bone marrow-derived dendritic cells (BMDCs) from BALB / c mice;

[0384] (2) BMDCs were cultured to day 5 and resuspended in 1640 complete medium. 40,000 cells were seeded per well in a 96-well plate. EM-NPs, TM-NPs, Mix NPs, and HM-NPs solutions with a final concentration of 1 mg / mL were added for co-incubation. Three parallel wells were set up in each group. An equal volume of 1640 complete medium was also added as a control group (Control).

[0385] (3) After incubation at 37°C for 24 h, cells were collected and washed three times with PBS;

[0386] (4) The cells were stained with FITC-CD11c, PE-CD80, and PE-CD86 flow cytometry antibodies for 20 minutes in the dark;

[0387] (5) BD-C6 flow cytometer was used to detect the levels of CD80 and CD86 expressed on the cell surface of each group, and 10,000 cells were collected from each group.

[0388] The results are as follows Figure 4 As shown, it can be seen that HM-NPs hybrid membrane nanoparticles have the strongest potential to stimulate DC cell maturation compared with other groups.

[0389] 3. Experiment on promoting IFN-γ secretion by mouse spleen lymphocytes:

[0390] (1) BALB / c mice were randomly divided into 5 groups, with 3 mice in each group;

[0391] (2) On days 1, 3, and 7, mice in each group were subcutaneously inoculated with EM-NPs, TM-NPs, Mix NPs, and HM-NPs vaccines (100 μg / mouse). An equal volume of PBS solution served as the control group (Control, Ctrl).

[0392] (3) On the 8th day, spleen cells from each group of mice were collected and ground, and then filtered through a 70 μm filter to obtain a mixed spleen lymphocyte suspension;

[0393] (4) The spleen lymphocytes of each group of mice were co-incubated with the tumor cell membranes of the same origin as those in the HM-NPs vaccine (as tumor antigens) in a 37°C 5% CO2 incubator for 24 h;

[0394] (5) The cells in each group were lysed and the IFN-γ secretion was detected using an ELISPOT (enzyme-linked immunospot) kit;

[0395] (6) Use CTL-ImmunoSpot Plate Reader software to count the number of spots in each group and analyze the data.

[0396] The results are as follows Figure 5 and Figure 6 As shown, it can be seen that HM-NPs hybrid membrane nanoparticles have the strongest potential to stimulate spleen lymphocytes to secrete IFN-γ compared with other groups.

[0397] Taken together, the above three experimental results indicate that HM-NPs hybrid membrane nanoparticles have improved potential to enhance innate and specific immunity.

[0398] Example 3

[0399] This example explores the effects of the four products prepared in Example 1 on stimulating the systemic immune system of mice to secrete inflammatory factors and chemokines. The specific steps are as follows:

[0400] (1) BALB / c mice were randomly divided into 5 groups, with 5 mice in each group;

[0401] (2) On days 1, 3, and 7, mice in each group were subcutaneously inoculated with EM-NPs, TM-NPs, Mix NPs, and HM-NPs vaccines (100 μg / mouse). An equal volume of saline was used as the control group (Control, Ctrl).

[0402] (3) Twelve hours after subcutaneous injection of the vaccine on day 7, serum was collected from mice in each group and the secretion levels of inflammatory factors and chemokines were detected using the ProcartaPlex multiplex immunoassay kit;

[0403] (4) Data were analyzed using a BD-C6 flow cytometer and processed using ProcartaPlex Analysis software.

[0404] The results are as follows Figure 7 As shown in Figure A-7M, the HM-NPs hybrid membrane nanoparticles have a certain effect on the secretion of inflammatory factors and chemokines by the systemic immune system. Compared with the blank control group, only the proinflammatory cytokines IL-6 and IL-1β increased significantly, while the other 11 inflammatory factors and chemokines remained basically unchanged. This indicates that the HM-NPs hybrid membrane nanovaccine has good biosafety.

[0405] Example 4

[0406] This example explores the effects of the four products prepared in Example 1 on lymph node enrichment in vivo, and the specific steps are as follows:

[0407] (1) Construction of membrane nanoparticles of each group encapsulating fluorescent dye IR780: The hydrophilic fluorescent dye IR780 was encapsulated in PLGA using the double emulsion method, specifically: 200 μL of 1 mg / mL hydrophilic fluorescent dye IR780 was added to 1 mL of 10 mg / mL PLGA dichloromethane solution, and the emulsion was carried out for 3 minutes; 1% surfactant sodium cholate was added and the emulsion was carried out for 5 minutes; the organic solvent dichloromethane was removed by rotary evaporation for 10 minutes in the dark; centrifugation was carried out at 11000 g for 15 minutes, and the PLGA nanoparticles encapsulating fluorescent dye IR780 were obtained. Then the PLGA nanoparticles were mixed with the membrane microparticles of each group prepared in advance. The specific steps and feeding ratios were the same as those in Example 1.

[0408] (2) BALB / c mice were randomly divided into 4 groups, with 4 mice in each group;

[0409] (3) Each group of mice was subcutaneously injected with fluorescently labeled membrane nanoparticles on the right back. Lymph nodes of each group of mice were collected 12 hours later and fluorescence imaging was performed using a small animal optical 3D living imaging system and analyzed using LivingImage software.

[0410] The results are as follows Figure 8 and Figure 9 As shown, it can be seen that the fluorescence signal of HM-NPs hybrid membrane nanoparticles in the lymph nodes is the strongest compared with other groups, indicating that HM-NPs hybrid membrane nanoparticles have a strong potential for lymph node enrichment.

[0411] Example 5

[0412] This example investigates the biosafety of the HM-NPs hybrid membrane nanoparticles prepared in Example 1 by using a hemolysis test. The specific steps are as follows:

[0413] (1) 1 mL of blood was collected from BALB / c mice, dissolved in 2 mL of PBS solution, and centrifuged at 3000 rpm for 10 min.

[0414] (2) Wash three times with PBS and gently pipette until the blood supernatant is colorless;

[0415] (3) The centrifuged red blood cells were diluted with 2 mL of PBS, and 0.1 mL of the blood dilution was added to 0.9 mL of the test sample (different concentrations of HM-NPs hybrid membrane nanoparticles, PBS as a negative control, and three times water as a positive control);

[0416] (4) All samples were placed on a shaker at 150 rpm and 37°C for 3 h;

[0417] (5) The shaken mixture was centrifuged at 12,000 rpm for 10 min and photographed. The absorbance of the supernatant at 541 nm was detected using an enzyme-labeled instrument to calculate the hemolysis rate.

[0418] The results are as follows Figure 10 As shown, it can be seen that after the HM-NPs hybrid membrane nanoparticles of various concentrations were mixed with red blood cells, no hemolysis occurred, indicating that the tumor vaccine involved in the present invention has good biosafety.

[0419] Example 6

[0420] This example investigates the effects of the four products prepared in Example 1 on inhibiting postoperative tumor recurrence in 4T1-luciferase breast cancer-bearing mice undergoing tumor resection. The experimental operation flow chart of this example is shown in FIG. Figure 11 The specific steps are as follows:

[0421] (1) Female BALB / c mice were randomly divided into 5 groups, with 6 mice in each group. 200,000 4T1-luciferase breast cancer cells were subcutaneously inoculated on the right back of each mouse to establish a mouse breast cancer model (day 0);

[0422] (2) Observe and record the tumor growth of mice every other day, and use a small animal fluorescence imaging system to detect the fluorescence intensity of tumors in tumor-bearing mice every other week. Figure 12 As shown;

[0423] (3) When the average tumor volume reaches 300 mm 3 Around , all mouse tumors were surgically removed, and the wounds were sutured (day 7);

[0424] (4) Grind and crush the cut tumor mass, digest the tumor tissue with a mixed solution of collagenase, DNAse and hyaluronidase at 37°C, filter the digested cell suspension through a 70μm filter and centrifuge, discard the supernatant after centrifugation, add extraction buffer and resuspend to obtain a cell suspension; use a cell disruptor to perform ice bath ultrasonic crushing on the cell suspension (35W, 5min); centrifuge the crushed cell suspension (3000g, 5min, 4°C); take the supernatant from the previous step and centrifuge again (10000g, 10min, 4°C); take the cell supernatant after centrifugation and add it to an ultracentrifuge tube for ultracentrifugation (100000g, 2h, 4°C); discard the supernatant after ultracentrifugation, resuspend with 1mL PBS to obtain tumor cell membrane fragments TM derived from surgical resection, and store at -80°C;

[0425] (5) Prepare various membrane nanoparticles according to the method in Example 1;

[0426] (6) Potassium luciferin was injected intraperitoneally into the mice after surgery, and small animal fluorescence imaging was performed to observe and count the residual tumor volume of the mice after surgery (day 9);

[0427] (7) The mice were randomly re-grouped according to the results of small animal fluorescence imaging into 5 groups, with 6 mice in each group;

[0428] (8) Each group of mice was subcutaneously injected with EM-NPs, TM-NPs, Mix NPs, and HM-NPs (100 μg / mouse) on days 10, 12, and 16, respectively. The saline group served as the control group (Control, Ctrl);

[0429] (9) Observe and record the tumor growth of mice every other day. When the tumor volume of mice reaches 1000mm 3 The mice were considered dead when the number of mice died was about 20. The specific time of death of each group of mice was recorded.

[0430] The results are as follows Figure 13-15 As shown, it can be seen that the HM-NPs hybrid membrane nanoparticles involved in the present invention have a more significant potential to inhibit tumor recurrence in mice after breast cancer surgery compared with other groups, with an inhibition rate of 83.3%.

[0431] Example 7

[0432] This example investigates the effects of the four products prepared in Example 1 on inhibiting tumor recurrence and tumor cell re-stimulation in CT26 colon cancer-bearing mice undergoing tumor resection. The experimental operation flow chart of this example is shown in FIG. Figure 16 The specific steps are as follows:

[0433] 1. Experiment on preventing recurrence of colon cancer after surgery:

[0434] (1) Male BALB / c mice were randomly divided into 5 groups, with 6 mice in each group. 200,000 CT26 colon cancer cells were subcutaneously inoculated on the right back of each mouse to establish a mouse colon cancer model (day 0);

[0435] (2) Observe and record the tumor growth of mice every other day;

[0436] (3) When the average tumor volume reaches 300 mm 3 Around 24 h, all mouse tumors were surgically removed, and the wounds were sutured (day 7);

[0437] (4) Grind and crush the cut tumor mass, digest the tumor tissue with a mixed solution of collagenase, DNAse and hyaluronidase at 37°C, filter the digested cell suspension through a 70μm filter and centrifuge, discard the supernatant after centrifugation, add extraction buffer and resuspend to obtain a cell suspension; use a cell disruptor to perform ice bath ultrasonic crushing on the cell suspension (35W, 5min); centrifuge the crushed cell suspension (3000g, 5min, 4°C); take the supernatant from the previous step and centrifuge again (10000g, 10min, 4°C); take the cell supernatant after centrifugation and add it to an ultracentrifuge tube for ultracentrifugation (100000g, 2h, 4°C); discard the supernatant after ultracentrifugation, resuspend with 1mL PBS to obtain tumor cell membrane fragments TM derived from surgical resection, and store at -80°C;

[0438] (5) Prepare various membrane nanoparticles according to the method in Example 1;

[0439] (6) The mice were randomly divided into 5 groups, with 15 mice in each group;

[0440] (7) Each group of mice was subcutaneously injected with EM-NPs, TM-NPs, Mix NPs, and HM-NPs (100 μg / mouse) on days 10, 12, and 16, respectively. The saline group served as the control group (Control, Ctrl);

[0441] (8) Observe and record the tumor growth of mice every other day. When the tumor volume of mice reaches 1000mm 3 The mice were considered dead when the number of mice died was about 20. The specific time of death of each group of mice was recorded.

[0442] The results are as follows Figure 17-18 As shown, it can be seen that the HM-NPs hybrid membrane nanoparticles involved in the present invention have a more significant potential to inhibit tumor recurrence in mice after colon cancer surgery compared with other groups, with an inhibition rate of 100%.

[0443] 2. Postoperative tumor re-challenge test:

[0444] (1) After 60 days, the mice vaccinated with HM-NPs vaccine in group 1 were randomly divided into three groups, with 5 mice in each group;

[0445] (2) One group of mice was subcutaneously inoculated with 200,000 4T1 breast cancer cells on the right back, another group of mice was subcutaneously inoculated with 200,000 CT26 colon cancer cells on the right back, and the remaining group of mice was not inoculated with tumor cells (inoculated with normal saline as a control);

[0446] (3) Observe and record the tumor growth of each group of mice every other day. When the tumor volume of the mice reaches 1000mm 3The mice were considered dead when the CT scan was taken, and the specific time of death of each group of mice was recorded.

[0447] The results are as follows Figure 19 , it can be seen that: when CT26 colon cancer cells "invaded" the mice for the second time, the mice in the hybrid membrane vaccine group that had been vaccinated with tumor cell membranes derived from colon cancer tissue had the ability to completely resist the tumor; and when 4T1 breast cancer cells "invaded" the mice, because the mice had not been exposed to 4T1 breast cancer cells before and had not obtained 4T1 breast cancer cell-related tumor antigens, they had no immunity to 4T1 breast cancer cells. The IL-6, TNF-α, IL-1β and IFN-γ levels in the serum of each group of mice were measured on the 90th day, and the results are as follows Figure 20 As shown in A-20D, it can be seen that the pro-inflammatory factors (IL-6, TNF-α and IL-1β) secreted by mice inoculated with CT26 colon cancer cells for the second time were significantly higher than those in the other two groups, and the IFN-γ secreted by them was also increased to a certain extent compared with the mice inoculated with 4T1 breast cancer cells.

[0448] Example 8

[0449] This example explores the dosage ratio of the Gram-negative bacterial inner membrane and the solid tumor cell membrane derived from surgical resection in the HM-NPs hybrid membrane nanoparticles involved in the present invention, specifically including the following contents:

[0450] (1) Hybrid membrane nanoparticles with different membrane protein concentration ratios were prepared according to the method of Example 1, specifically: Gram-negative bacterial inner membrane EM alone, surgical resection-derived solid tumor cell membrane TM alone, and hybrid membranes with a mass ratio of Gram-negative bacterial inner membrane EM to surgical resection-derived solid tumor cell membrane TM of 1:100, 1:75, 1:50, 1:25, 1:10, 1:5, 1:3, 1:1, 1:0, 0:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1, or 100:1, and an equal volume of PBS solution was used as a control group (Control);

[0451] (2) Bone marrow dendritic cells (BMDCs) from BALB / c mice were extracted and cultured according to the method of Example 2. BMDCs were cultured to day 5, resuspended in 1640 complete medium, and divided equally into 6 portions, 50,000 cells per well, and seeded into 96-well plates. Hybrid membrane nanoparticles with different membrane protein ratios were added for co-incubation, with 3 replicates per group.

[0452] (3) After incubation at 37°C for 24 h, the culture supernatant of each group was removed and the levels of IL-6, TNF-α, and IL-1β in the supernatant were detected using an Elisa kit.

[0453] The results are as follows Figure 21 As shown in A-21C, it can be seen that compared with other groups, the hybrid membrane with an EM:TM ratio of 3:1 has the strongest ability to stimulate proinflammatory cytokines (IL-1β, TNF-α, IL-6), indicating that it has the strongest potential to stimulate the body's innate immunity.

[0454] Example 9

[0455] This example prepares a membrane nanoparticle system consisting of nanoparticles (hereinafter referred to as HM-NPs-2) formed from a hybrid membrane composed of the inner membrane of Klebsiella pneumoniae (CG43 strain) and the cell membrane of HepG2 liver cancer tissue, encapsulated in a zirconium (Zr)-based MOF (metal-organic framework). The preparation method comprises the following steps:

[0456] (1) Extracting the inner membrane EM of Klebsiella pneumoniae, the specific operation is the same as that in Example 1, including: culturing and amplifying the bacteria; adding lysozyme to rupture the cell wall and low-speed centrifugation to obtain protoplasts; adding extraction buffer and finally obtaining the inner membrane EM of Klebsiella pneumoniae by density gradient centrifugation;

[0457] (2) Extracting cell membranes TM from HepG2 liver cancer tissue. The specific operation is the same as that in Example 1, including: subcutaneous implantation of tumors in BALB / c mice; surgical excision when the tumors grow to a certain size; chopping with scissors, adding three enzymes to digest and lyse the tumor tissue, grinding through a 70 μm filter; adding extraction buffer and disrupting the cells using an ultrasonic cell disruptor; and finally centrifuging to obtain liver cancer cell membranes derived from the liver cancer tissue.

[0458] (3) 3 mg of extracted Pneumococcus endomembrane EM and 1 mg of extracted surgically removed tumor cell membrane TM (with the same membrane protein concentration) were mixed in PBS and shaken at 37°C for 15 min. The mixed membrane solution was then passed through a liposome extruder with a filter membrane pore size of 400 nm and extruded back and forth 13 times (one back and forth counted as one time) to obtain HM hybrid membrane microparticles.

[0459] (4) Synthesis of MOF: 300 mg ZrOCl2·8H2O, 100 mg H2TCPP and 2.8 mg benzoic acid (BA) were dissolved in 100 mL dimethylformamide (DMF) solvent and stirred at 90 °C for 5 h under nitrogen protection. After cooling to 25 °C, the MOF product was collected by centrifugation at 10,000 g for 30 min and washed three times with DMF. The synthesized MOF was dissolved in DMF and stored at 4 °C. It needed to be washed three times with deionized water before use in the experiment.

[0460] (5) 50 μL of the 2 mg / mL hybrid membrane microparticles prepared in step (3) was mixed with 500 μL of the 1 mg / mL MOF prepared in step (4), and the mixture was sonicated in a bath sonicator (ultrasonic water bath) for 30 min. The product was collected by centrifugation to obtain HM-NPs-2 hybrid membrane nanoparticles.

[0461] Example 10

[0462] This example prepares a membrane nanoparticle system for Brucella PBP 39 The preparation method of the nanoparticle system (hereinafter referred to as HM-NPs-3) formed by the hybrid membrane composed of the endometrium and the cell membrane of the SN12-PM6SN12-PM6 renal cancer tissue cells encapsulating MSN (mesoporous silica) is as follows:

[0463] (1) Extracting Brucella inner membrane EM, the specific operation is the same as Example 1, including: bacterial culture and amplification; adding lysozyme to rupture the cell wall and low-speed centrifugation to obtain protoplasts; adding extraction buffer and finally obtaining Brucella inner membrane EM by density gradient centrifugation;

[0464] (2) Extracting cell membranes TM from SN12-PM6 renal cancer tissue. The specific procedures were the same as those in Example 1, including: subcutaneous implantation of tumors in BALB / c mice; surgical resection when the tumors grew to a certain size; chopping the tumor tissue with scissors, adding three enzymes to digest and lyse the tumor tissue, and grinding it through a 70 μm filter; adding extraction buffer and disrupting the cells using an ultrasonic cell disruptor; and finally centrifuging to obtain liver cancer cell membranes derived from the liver cancer tissue.

[0465] (3) 3 mg of extracted Brucella inner membrane EM and 1 mg of extracted surgically removed tumor cell membrane TM (with the same membrane protein concentration) were mixed in PBS and shaken at 37°C for 15 min. The mixed membrane solution was then passed through a liposome extruder with a filter membrane pore size of 400 nm for a total of 13 times (one back and forth counted as one time) to obtain HM hybrid membrane microparticles;

[0466] (4) Synthesis of MSNs: 1.5 mL of tetraethyl orthosilicate (TEOS) and 5 mL of acetone were used as the oil phase, followed by the addition of 40 mL of an aqueous solution containing 0.15 g of hexadecyltrimethylammonium bromide (CTAB); the mixture was stirred and sheared at 10,000 rpm for 5 min to form an emulsion; 50 μL of ammonia water was added to the emulsion, followed by magnetic stirring at 400 rpm for 2 h to evaporate the organic solvent; the suspension was centrifuged, and the precipitate was washed three times with deionized water and then freeze-dried; tetraethyl orthosilicate (TEOS) was hydrolyzed and concentrated at the oil / water interface to form a silica shell; finally, the droplets were removed by evaporation (volatilization) during the reaction or drying process to obtain mesoporous silica MSNs.

[0467] (5) 50 μL of 2 mg / mL HM hybrid membrane microparticles prepared in step (3) was mixed with 500 μL of 1 mg / mL MSN prepared in step (4), and the mixture was sonicated in a bath sonicator (ultrasonic water bath) for 30 min. The product was collected by centrifugation to obtain HM-NPs-3 hybrid membrane nanoparticles.

[0468] Example 11

[0469] This example explores the effects of the two products prepared in Example 9 and Example 10 on enhancing innate immunity and specific immunity, and the specific contents include:

[0470] 1. Experiment on promoting BMDC to secrete pro-inflammatory cytokines:

[0471] For specific operation methods, please refer to Example 2.

[0472] The results showed that HM-NPs-2 and HM-NPs-3 hybrid membrane nanoparticles promoted the secretion of IL-6, TNF-α, and IL-1β by BMDCs at levels similar to those of HM-NPs, and had significant potential to enhance the body's innate immunity.

[0473] 2. Experiments to promote BMDC maturation:

[0474] For specific operation methods, please refer to Example 2.

[0475] The results showed that HM-NPs-2 and HM-NPs-3 hybrid membrane nanoparticles promoted the expression of CD80 and CD86 on the surface of BMDC cells at levels similar to those of HM-NPs, and had significant potential to stimulate DC cell maturation.

[0476] 3. Experiment on promoting IFN-γ secretion by mouse spleen lymphocytes:

[0477] For specific operation methods, please refer to Example 2.

[0478] The results showed that the number of spots of the HM-NPs-2 and HM-NPs-3 hybrid membrane nanoparticle groups was similar to that of HM-NPs, and they had significant potential to stimulate IFN-γ secretion by splenic lymphocytes.

[0479] The combined results of the above three experiments indicate that HM-NPs-2 and HM-NPs-3 hybrid membrane nanoparticles have significant potential to enhance innate and specific immunity.

[0480] Example 12

[0481] Study plan

[0482] The Escherichia coli inner membrane and tumor cell membrane of the resected autologous tumor tissue were separated, and four batches of EM and TM samples were subjected to whole proteomic analysis using liquid chromatography-mass spectrometry (LC-MS). PLGA polymer nanoparticles (NP) were prepared using the double emulsion method. Bone marrow-derived dendritic cells (BMDC) were induced to differentiate from mouse bone marrow mesenchymal stem cells, which were freshly extracted from 6-8 week old female C57BL / 6J mice. This application studies the innate immune stimulation response and specific immune stimulation response of different membrane nanoparticles in vivo and in vitro, the physicochemical properties of different membrane nanoparticles, and the proteomic characterization. For animal experiments, the tumor volume was measured using an electronic vernier caliper. When the volume of the tumor reached approximately 300mm 3 The mice were then immunized with different vaccine formulations on days 3, 5, and 9 after surgery. For the CT-26 tumor re-challenge model, mice from the HM-NP vaccination group were randomly divided into three groups and vaccinated with CT-26 colon adenocarcinoma cells or 4T1 breast cancer cells 60 days later. In the immune cell depletion model, one day before the start of HM-NP treatment, depletion antibodies targeting designated surface markers were injected intraperitoneally to deplete macrophages (CSF1R antibody), NK cells (ASGM1 antibody), CD8 + T cells (CD8 antibodies) or CD4 + T cells (CD4 antibody). Except for the untreated group, all mice were inoculated with HM-NP on days 3, 5, and 9 after surgery. To evaluate in vivo biosafety, at the endpoint of the tumor regression experiment in the 4T1-Luc model, hematoxylin-eosin (H&E)-stained sections of the main organs of each group of mice were performed, and liver and kidney function indicators in the serum of each group of mice were measured.

[0483] In vitro BMDC uptake assay of HM-NPs

[0484] Fluorescently labeled nanoparticles were prepared by incorporating rhodamine B into the PLGA core during synthesis. BMDCs were generated by induced differentiation from fresh bone marrow mesenchymal stem cells from C57BL / 6J mice. The cells were cultured in RPMI 1640 medium containing 10% FBS and penicillin-streptomycin. The cells were added to a 1.5 mL centrifuge tube (40,000 cells / tube) and a final concentration of 0.4 mg mL was added. -1Different vaccine formulations were co-cultured at 37°C for 8 hours. Cells were then harvested by centrifugation at 500 × g for 3 minutes and resuspended in FACS buffer (RPMI 1640 medium supplemented with 2% FBS). Fluorescence signals were assessed using a BD D Accuri C6 FACS flow cytometer (BD Biosciences, San Jose, USA) and analyzed using BD D Accuri C6 software.

[0485] Confocal microscopy detection of HM-NPs

[0486] To evaluate the process of internalization into dendritic cells, membrane-coated NPs and BMDCs with Cy5.5-labeled fluorescent lysosomes were prepared. EM-NPs, TM-NPs, and HM-NPs were co-incubated with BMDCs and imaged under a confocal laser scanning microscope (Zeiss 710, Zeiss Microsystems, Germany). To further compare the colocalization efficiency of HM-NPs and Mix NPs, BMDCs were treated with HM-NPs or Mix NPs and imaged under a confocal laser scanning microscope. EM-NPs were labeled with a primary antibody specific for the bacterial inner membrane, FtsZ, and a secondary goat antibody against rabbit IgG (Alexa Fluor@633 goat, Abcam, Cambridge, UK). + / K + -ATPase and goat secondary antibodies against mouse IgG (Alexa Fluor@488 goat, Abcam, Cambridge, UK) were used to label TM-NPs.

[0487] In vitro innate immunity assays

[0488] To investigate the ability of different membrane NPs to stimulate innate immune responses in vitro, BMDCs were added to 96-well plates (40,000 cells / well) and incubated with a final concentration of 0.4 mg mL -1 The different nanoformulations were incubated together at 37 °C for 24 h. The supernatants were collected and analyzed for the concentrations of IL-6, TNF-α, and IL-1β using the corresponding specific ELISA kits.

[0489] In vitro and in vivo DC maturation assays

[0490] To investigate the ability of different membrane NPs to promote BMDC maturation in vitro, BMDCs were added to 1.5 mL centrifuge tubes (40,000 cells / tube) and incubated with a final concentration of 0.4 mg mL -1The different nanoformulations were co-incubated at 37°C for 24 hours. The cells were then collected by centrifugation and resuspended in FACS buffer. FITC-CD11c, PE-CD80, and PE-CD86 were used for fluorescent labeling and analyzed using a BD Accuri C6 FACS flow cytometer and BD Accuri C6 software. To evaluate DC maturation in vivo, female BALB / c mice were subcutaneously inoculated with 4T1 breast cancer cells. When the tumor volume reached approximately 300 mm 3 The mice were randomly divided into five groups (n=5 mice per group) after surgery. The mice were immunized with different vaccine preparations on days 3, 5, and 9 after surgery.

[0491] T cell response measurement

[0492] To examine whether HM-NPs can effectively activate T cells, female BALB / c mice were subcutaneously inoculated with 4T1 breast cancer cells. The surgical procedures and vaccination schedule were identical to those in the study protocol described above. Splenocytes were collected 12 hours after the last vaccination and incubated with 4T1 cell membranes (as antigens) for at least 24 hours. ELISPOT kits pre-coated with IFN-γ capture antibodies were used to assess T cell IFN-γ release induced by the different membrane NPs. Plates were scanned using a CTL-ImmunoSpot plate reader, and data were analyzed using CTL ImmunoSpot software.

[0493] Serum cytokine assay

[0494] To assess the extent of systemic inflammatory responses after treatment with different vaccine formulations, female BALB / c mice were subcutaneously inoculated with 4T1 breast cancer cells. Surgical and vaccination procedures were performed as described above. Serum inflammatory cytokines and chemokines were measured using Luminex magnetic bead-based ELISA kits according to the manufacturer's instructions, using one-fifth the recommended amounts of capture beads and detection antibodies.

[0495] Tumor regression experiment

[0496] Female BALB / c or C57BL / 6 mice were subcutaneously inoculated with 4T1-Luc, CT26, B16F10, or EMT-6 tumor cells (2 × 10 per mouse). 5 Surgical and vaccination procedures were performed as described above. An IVIS (Spectrum CT, PerkinElmer, UK) was used to monitor the bioluminescent signal produced by 4T1-Luc cells. The volume of all tumors was measured every other day using an electronic caliper. Tumor volume was calculated according to the following formula:

[0497] V = (L × W × W) / 2 (L, longest dimension; W, shortest dimension)

[0498] CT-26 Tumor and Rechallenge Model

[0499] To establish a CT-26 tumor rechallenge model, mice treated with HM-NPs were randomly divided into three groups 60 days after surgery and inoculated with either CT-26 colon adenocarcinoma cells or 4T1 breast tumor cells (n=12). Tumor volumes were measured every other day using an electronic caliper. Cytokine secretion in the serum of rechallenged mice was measured using specific ELISA kits.

[0500] In vivo macrophages, NK cells, CD8 + T cells or CD4 + T cell depletion assay

[0501] Female BALB / c mice were subcutaneously inoculated with CT-26 colon adenocarcinoma cells (2×10 5 The surgical procedure and vaccination schedule were the same as described above. Intraperitoneal injection of antibodies depleted immune cell subsets. This started one day before the start of HM-NP vaccine treatment. The types and usage of immune cell depletion antibodies were as follows: macrophages with anti-mouse CSF1R (CD115, BioXCell; 300 μg injected every other day), NK cells with anti-ASGM1 (anti-mouse Asialo-GM1, BioLegend; 50 μL / injection, twice a week), CD8 + T cells (Lyt 2.1, BioXCell; 400 μg / injection, twice a week) and CD4 + T cells (GK1.5, BioXCell; 200 μg / injection, twice a week). The volume of all tumors was measured every other day using an electronic caliper. The presence of macrophages, NK cells, CD8 T cells, and NK cells was confirmed by flow cytometry of PBMCs. + T cells and CD4 + Cell depletion of T cells.

[0502] Statistical analysis

[0503] Statistical analysis was performed using GraphPad Prism software version 5.0. Differences between the two experimental groups were assessed by one-way analysis of variance or two-way analysis of variance using Bonferroni's multiple comparison test. Heat maps of the Luminex-based ProcartaPlex multiple immunoassay were analyzed in groups by two-way analysis of variance with Bonferroni's multiple comparison test. Survival curves were evaluated using the log-rank (Mantel-Cox) test. Data are presented as mean ± standard deviation, as indicated in the figure legends. Statistical significance was set as follows: *p < 0.05. **, p < 0.01; ***, p < 0.001, ****, p < 0.0001, and ns indicates no significant difference.

[0504] Sources

[0505] Poly(lactide-co-glycolic acid)-OH (75:25, relative molecular mass [Mr] 20,000 Da) was purchased from Jinandaigang Biotechnology Company (Beijing, China). Phosphate-buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, and fetal bovine serum (FBS) were purchased from Wisent Bio-Products (Montreal, Canada). BCA protein assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Pre-coated ELISA kit for LPS detection was purchased from eBioscience (San Diego, CA). Anti-FtsZ rabbit antibody was purchased from Agrisera ( Sweden). Mouse anti-Na + / K +Antibodies against α-ATPase, ATP5A (IM CVa), ubiquinol-cytochrome c reductase core protein I (IMCore I), VDAC1 / porin (OM Porin), matrix cyclophilin D (Matrix CypD), and IMS cytochrome c (IMS) (Cytc) were purchased from Abcam (Cambridge, UK). Anti-rabbit IgG-gold conjugate (gold nanoparticle size, 5 nm) and anti-mouse IgG-gold conjugate (gold nanoparticle size, 10 nm) were purchased from Abcam (Cambridge, UK). Murine IL-4 and GM-CSF were purchased from Beyotime Biotechnology (Shanghai, China). ELISA kits for IL-6, TNF-α, IL-1β, and IFN-γ analysis were purchased from eBioscience (San Diego, CA). Mouse antibodies against FITC-CD11c, PE-CD80, and PE-CD86 were purchased from BioLegend (San Diego, USA). A pre-coated ELISPOT kit for IFN-γ was purchased from Dakwei Biotechnology Co., Ltd. (Beijing, China). Luminex bead-based ELISA kits were purchased from Thermo Fisher Scientific (Waltham, MA, USA). D-luciferin potassium was purchased from bide Pharmatech Co. Ltd (Shanghai, China). Macrophages (CD115, anti-mouse CSF1R), CD8 + T cells (Lyt 2.1, anti-mouse CD8) and CD4 + T cell depletion antibodies (GK1.5, anti-mouse CD4) were purchased from Bio X Cell, Inc., USA. NK cell depletion antibodies (anti-mouse Asialo-GM1) were purchased from BioLegend (San Diego, USA). Unless otherwise stated, other chemicals were purchased from Wallead Technology Co., Ltd. (Beijing, China).

[0506] Cell lines and animals

[0507] Mouse 4T1 breast cancer cells, 4T1-luciferase breast cancer cells, CT-26 colon tumor cells, EMT-6 breast cancer cells, and B16F10 melanoma cells were purchased from the American Type Culture Collection (ATCC, Manassas, USA). -1 Glucose and 0.11 g·L -1 4T1, 4T1-Luc, EMT-6, and B16F10 cells were cultured in RPMI 1640 medium supplemented with sodium pyruvate. CT-26 cells were maintained in DMEM supplemented with 10% FBS. All cells were incubated at 37°C in 5% CO2.

[0508] Female BALB / c or C57BL / 6 mice were purchased from Weitonglihua Laboratory Animal Technology Co., Ltd. (Beijing, China) and maintained under pathogen-free conditions. All animal experiments were performed under the guidance of the Institutional Animal Care and Use Committee (IACUC) of the National Center for Nanoscience and Technology, Chinese Academy of Sciences (Beijing, China).

[0509] Generation of bone marrow-derived dendritic cells (BMDCs)

[0510] BMDCs were generated from BMSCs of female BALB / c mice (6 to 8 weeks old) by inducing differentiation. Briefly, after euthanasia of the mice, the bones were collected and placed in 70% ethanol and then washed with PBS. The two distal ends of the bones were cut open and the bone marrow was collected by gently washing with RPMI 1640 medium. The red blood cells in the collected cells were lysed with ACK buffer and the remaining cells were collected by centrifugation at 300 × g for 5 minutes and then cultured in RPMI 1640 medium in 6-well plates (1 × 10 6 cells / well). 10% FBS, 2 mM L-glutamine, 100 μg mL -1 Penicillin, 100 μg mL -1 Streptomycin, 0.05 mM β-mercaptoethanol (β-ME), 10 ng mL -1 Mouse IL-4 and 20 ng mL -1 Fresh medium was added on days 2 and 4. On day 6, non-adherent cells were aspirated and incubated with fresh medium in 6-well plates for further experiments.

[0511] Isolation of Escherichia coli-derived plasma membrane (EM)

[0512] The cytoplasmic membrane was isolated from the Escherichia coli strain DH5α. Briefly, the frozen E. coli DH5α cells were cultured in liquid LB medium at 37°C with shaking at 200 rpm overnight. When the OD600 was 1.2, the bacteria were collected by centrifugation (3000×g, 20 min, 4°C) and washed 3 times with PBS. The cell pellet was resuspended in 10 mL of buffer A (1 M sucrose, 0.2 M Tris-HCl, pH 8.0). Lysozyme was then added to a final concentration of 2 mg mL -1 The cells were incubated at 37°C with shaking at 120 rpm for 1 hour. -1) in sterile water (90 mL) and gently mix the tube 20 times. Protoplasts were collected by centrifugation at 3,000 × g for 20 minutes at 4°C and resuspended in 10 mL of ice-cold buffer B (20 mM Tris-HCl, pH 7.2, 50 mM NaCl, 5 mM EDTA) containing 20% ​​w / v sucrose. The cells were lysed with sucrose. The lysate was cleared of cell debris by centrifugation at 10,000 × g for 30 minutes at 4°C. A sample (5 mL) of the supernatant was placed on a discontinuous sucrose gradient (bottom to top: 10 mL at 50%, 5 mL at 46%, 10 mL at 42%, 10 mL at 36%, 5 mL at 32%) and 10 mL at 27%) solution) and centrifuged at 113,000 × g for 2 hours at 4°C in a Beckman Optima XPN-100 ultracentrifuge (Brea, CA, USA) using a 70Ti rotor. Subsequently, 1.5 mL of the cytoplasmic membrane-containing fraction was extracted and diluted with ice-cold buffer B to reduce the sucrose concentration to 10% (w / v). The membranes were collected by centrifugation at 113,000 × g for 1 hour at 4°C, resuspended in PBS, and stored at -80°C for subsequent experiments. The protein concentration of the final membrane preparation was quantified using a BCA protein assay kit. To analyze the protein composition of EM, proteomic analysis was performed (H Wayen Biotechnology Co., Ltd., Shanghai, China), and a total of 2,302 proteins were detected.

[0513] Isolation of Tumor Cell Membranes(TM) from Resected Tumor Tissue

[0514] To obtain cell membranes from tumor tissue, female BALB / c mice were subcutaneously inoculated with murine 4T1 breast cancer cells, 4T1-Luc breast cancer cells, or CT-26 colon cancer cells. On day 7 after tumor inoculation, the tumors (approximately 300 mm 3 ) were excised and cut into small pieces, and then treated with GBSS buffer (2 mL) containing collagenase IV (1.0 mg mL -1 ), DNase (0.1 mg mL -1 ) and hyaluronidase (0.1 mg mL -1) digested the tissue at 37 ° C. The cells were scraped and collected by centrifugation at 1000 × g for 5 minutes. The precipitate was homogenized in 2 mL of separation buffer (225 mM mannitol, 75 mM sucrose, 0.5% BSA, 0.5 mM EGTA, 30 mMTris and phosphatase and protease inhibitor cocktail) and then distributed in an ultrasonic bath (30 W) for 3 minutes in ice to fully destroy the cells. The cell homogenate was centrifuged at 3000 × g for 5 minutes at 4 ° C, and the supernatant was collected. The supernatant was further centrifuged at 10,000 × g for 10 minutes at 4 ° C, and the resulting supernatant was ultracentrifuged at 100,000 × g for 2 hours at 4 ° C. Finally, the cell membranes derived from tumor tissue were resuspended in 5 mM Bis-Tris buffer (pH 6.0) and stored at -80 ° C. The protein concentration of the membrane extract was determined using a BCA protein assay kit. To analyze the protein composition of TMs, proteomic analysis was performed (H Wayen Biotechnology Co., Ltd., Shanghai, China), and a total of 5,353 proteins were detected.

[0515] Production and physicochemical properties of HM-NPs

[0516] To prepare hybrid membrane (HM) vesicles, the mixed EM and TM were gently shaken for 15 min at 37 °C using a dry bath incubator before membrane fusion. HM vesicles were formed by repeated physical extrusion using a 400 nm cutoff extruder (Hamilton Company, Reno, Nevada, USA). Poly(lactide-co-glycolic acid)-OH (PLGA) nanoparticles were prepared using a double emulsion method. Briefly, PLGA was diluted to 10 mg mL -1 The concentration of 1% PLGA was dissolved in dichloromethane. Then, 1 mL of PLGA solution was mixed with 0.2 mL of sterile water. The mixture was emulsified by ultrasonic treatment (25W) in an ice bath for 3 minutes, and then 2 mL of 1% sodium cholate was added and emulsified by ultrasonic treatment (30W) in an ice bath for 5 minutes. The emulsion was added dropwise to 10 mL of 0.5% sodium cholate solution and stirred at room temperature for 30 minutes. After rotary evaporation for 15 minutes, PLGA NPs were harvested by centrifuging the emulsion at 10,000 × g for 15 minutes and washing twice in sterile water. To engineer HM-NPs, PLGA NPs and HM vesicles were repeatedly co-extruded again with a 200:1 cutoff extruder (Hamilton Company) at a polymer to membrane protein mass ratio of 5:1.

[0517] In order to examine the morphology of nanoparticles and vesicles, 10 μL samples were deposited on a carbon-coated copper grid and incubated for 15 minutes. Filter paper was used to absorb the residual liquid, and the sample on the grid was negatively stained for 8 minutes with 1% (v / v) uranyl acetate, then air-dried to measure the hydrodynamic particle size distribution, zeta potential, and polydispersity index (PDI) using a Zetasizer Nano ZS dynamic light scattering (DLS) instrument (Malvern, UK). Similar preparation methods were used to produce EM-NP and TM-NP.

[0518] Protein characterization of HM-NPs

[0519] To investigate the protein profile of HM-NPs, SDS-PAGE was performed and the resulting gels were stained with Coomassie Brilliant Blue (Solarbio, Beijing, China). All samples were prepared in loading buffer (Invitrogen, Carlsbad, CA, USA) and equal amounts of protein (10 μg) were loaded on NuPAGE Novex 12% separating gels (Invitrogen) in MOPAGE running buffer (Invitrogen). Western blot analysis was performed to identify the presence of specific protein markers for each component. Proteins were resolved as described above and transferred to nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA, USA) and probed with a primary antibody specific for FtsZ for characterization of the bacterial cytoplasmic membrane. Mouse anti-Na + / K + Antibodies to ATPase, ATP5A (IM CVa), ubiquinol-cytochrome c reductase core protein I (IM Core I), VDAC1 / Porin (OM Porin), matrix cyclophilin D (Matrix CypD), and IMS cytochrome c (IMS Cytc) were also used for detection; these are markers of tumor-derived membranes. For FtsZ, the secondary antibody used was an HRP-conjugated antibody against rabbit IgG, while for the other primary antibodies, the secondary antibody was an HRP-conjugated antibody against mouse IgG. To further characterize the HM-NPs, immunogold staining was performed. The samples were deposited onto carbon-coated copper grids. The grids were blocked with 1 wt% bovine serum albumin (BSA) in PBS. The EM-NP grids were incubated with 10 μL of anti-rabbit FtsZ (0.5 mg mL -1 ) staining, TM-NP grids were stained with 10 μL anti-mouse Na + / K + -ATPase (0.5 mg mL -1) staining. After incubation for one hour, the samples were washed 6 times in 1% BSA. The EM-NPs grids were then stained with 10 μL anti-rabbit IgG-gold conjugate (gold nanoparticle size of 5 nm) diluted 1:20 with 1% BSA. The TM-NP grids were stained with 10 μL anti-mouse IgG-gold conjugate (gold nanoparticle size of 10 nm) diluted 1:20 with 1% BSA. After incubation in the dark for one hour, the grids were washed 8 times with PBS. The samples were then fixed in 50 μL 1% glutaraldehyde in PBS and washed 8 times with sterile water for 2 minutes each. Finally, the sample grids were stained with 10 μL 1% uranyl acetate and examined using TEM (HT7700, HITACHI, Japan). The HM-NP grids were prepared according to the same procedure. Targeting FtsZ and Na + / K + A mixture of antibodies against ATPase was used as the primary antibody, and anti-rabbit IgG-gold conjugate (gold nanoparticle size of 5 nm) and anti-mouse IgG-gold conjugate (gold nanoparticle size of 10 nm) were combined to label HM-NPs.

[0520] Design and characterization of HM-NPs

[0521] The engineering of HM-NPs involves three steps: 1) preparation of EM-TM hybrid membrane (HM) vesicles, 2) synthesis of poly(lactide-glycolic acid) polymer nanoparticles (PLGA NPs), and 3) coating of HM onto PLGA NPs ( Figure 29A EM was isolated from Escherichia coli strain DH5α, and TM was isolated from autologous tumor cell membranes. The protein expression profiles of the E. coli inner membrane (Table 1A-1B) and the plasma membrane of surgically resected 4T1 tumor cells (Table 2A-2B) were characterized by liquid chromatography-mass spectrometry (LC-MS). Figure 22 The Venn diagram in EM( Figure 22 Left; E1, E2, E3 and E4) and TM( Figure 22 Right; T1, T2, T3 and T4) showed little difference in membrane protein between four different batches of samples. Membranes with different ratios were fused at a protein mass ratio of 1:100, 1:75, 1:50, 1:25, 1:10, 1:5, 1:3, 1:1, 1:0, 0:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1 or 100:1 between EM and TM, and HM was prepared by repeatedly passing through a liposome extruder with a 400 nm filter membrane pore size. To determine the optimal membrane ratio for activating DCs, HM with different membrane ratios were incubated with BMDCs for 24 hours, and the released pro-inflammatory cytokines were measured. The results showed that as the amount of EM increased, the release of pro-inflammatory cytokines from BMDCs increased ( Figures 23A-23C The group with a protein mass ratio of EM to TM of 3:1 induced the highest level of pro-inflammatory cytokine production.

[0522] Table 1A Protein expression profile of the plasma membrane of Escherichia coli DH5α identified by LC-MS

[0523]

[0524] Table 1B Protein expression profiles in the inner membrane of Gram-negative bacteria identified by LC-MS

[0525]

[0526]

[0527]

[0528] Proteins were analyzed in EM using Proteome Discoverer 2.4 software (Thermo Fisher Scientific, USA). Accession Number (Protein Accession Number): By default, displays the unique identifier assigned to the protein by the FASTA database used to generate the report. Abundance: Displays the sample abundance value before scaling and normalization. Data are presented as the mean ± standard deviation of four biologically independent plasma membrane samples from Escherichia coli DH5α.

[0529] By mass spectrometry analysis, some characteristic proteins were also identified, which can be used to confirm that the drug combination contains components derived from the bacterial inner membrane. For example, these bacterial inner membrane characteristic proteins can include proteins or functionally active fragments thereof selected from the following groups: cell division protein FtsZ, inner membrane protein YhcB, inner membrane protein translocation enzyme YidC, cell division protein NlpI, ABC transporter MsbA, inner membrane transport protein TatA, intermembrane phospholipid transport system lipoprotein MlaA, inner membrane protein TolQ, intermembrane transport lipoprotein PqiC, outer membrane protein TolC, outer membrane introduction protein FimD, outer membrane pore protein OmpC, intermembrane transport protein PqiB, major outer membrane lipoprotein Lpp, membrane-bound hemolytic wall protein transglycosylase MltB, UPF0194 membrane protein YbhG, putative membrane protein IgaA homolog, Transmembrane phospholipid transport system lipoprotein MlaA, inner membrane protein YejM, membrane-bound hemolytic wall protein transglycosylase MltA, transmembrane phospholipid transport system binding protein MlaC, inner membrane protein YlaC, transmembrane transport protein YebT, membrane-bound hemolytic murein transglycosylase MltC, transmembrane phospholipid transport system ATP binding protein MlaF, intermembrane phospholipid transport system binding protein MlaD, inner membrane protein YqjE, UPF0053 inner membrane protein YfjD, UPF0053 inner membrane protein YoaE, inner membrane type lytic murein transglycosylase A, UPF0394 inner membrane protein YedE, intermembrane phospholipid transport system binding protein MlaB, inner membrane protein YccF, transmembrane Phospholipid transport system permease protein MalE, inner membrane protein YedI, inner membrane protein YgaP, intermembrane transport protein PqiA, UPF0056 membrane protein YhcE, inner membrane protein YbhL, inner membrane protein YhjD, inner membrane transport protein YbaT, inner membrane protein YjjP, inner membrane protein YhaH, inner membrane protein YbjJ, inner membrane transport protein YqeG, UPF0053 inner membrane protein YtfL, arsenic pump membrane protein ArsB, inner membrane protein YpjD, membrane-bound lysozyme inhibitor MliC of C-type lysozyme, UPF0283 membrane protein YcjF, UPF0259 membrane protein YciC, inner membrane protein YgfX, inner membrane protein YbbJ, lipoprotein release system Transmembrane protein LolE, inner membrane protein YjcH, protein export membrane protein SecG, inner membrane protein YfdC, UPF0324 inner membrane protein YeiH, UPF0266 membrane protein YobD, TVP38 / TMEM64 family inner membrane protein YdjZ, membrane-associated protein UidC, inner membrane protein YbiR, inner membrane protein YhiM, membrane transport protein YfcA, inner membrane protein YdgK, membrane-bound hemolytic wall protein transglycosylase F, multidrug resistance outer membrane protein MdtP, UPF0208 membrane protein YfbV, peptidoglycan-associated lipoprotein, lipoprotein YiaD, lipoprotein YeaY, apolipoprotein N-acyltransferase, D-methionine-binding lipoprotein MetQ,Lipoprotein GfcD, lipoprotein YbjP, lipoprotein YgeR, lipoprotein release system ATP binding protein LolD, osmotic-inducible lipoprotein OsmE, osmotic-inducible lipoprotein OsmB, lipoprotein YdcL, lipoprotein YajG, lipoprotein NlpE, phosphatidylglycerol-lipoprotein diacylglycerol transferase, lipoprotein YghJ, lipoprotein YedD, lipoprotein YifL, lipoprotein YgdI, lipoprotein YbaY, lipoprotein signal peptidase, lipoprotein YceB, lipoprotein YajI and inner membrane protein YebE, and any combination thereof.

[0530] Table 2A Protein expression profiles of tumor membranes derived from surgically resected tumors identified by LC-MS

[0531]

[0532] Table 2B Protein expression profiles in tumor tissue-derived cell membranes identified by LC-MS

[0533]

[0534]

[0535]

[0536]

[0537] Proteins in the TM were analyzed using Proteome Discoverer 2.4 software (Thermo Fisher Scientific, USA). Protein accession number: By default, the unique identifier assigned to the protein by the FASTA database used to generate the report is displayed. Abundance: Displays the abundance value of the sample before scaling and normalization. Data are expressed as the mean ± standard deviation of 4 biologically independent tumor membrane samples from surgically resected tumors.

[0538] Through mass spectrometry analysis, some characteristic proteins were also identified, which can be used to confirm that the drug combination contains components derived from other organisms. For example, these characteristic proteins of other organisms can include proteins or functionally active fragments selected from the following groups: antigen peptide transporter 1, H-2 class II histocompatibility antigen γ chain, tyrosine protein kinase SYK, high affinity immunoglobulin epsilon receptor subunit γ, Ras-related C3 botulinum toxin substrate 2, tyrosine protein kinase BTK, receptor-type tyrosine protein phosphatase C, Na + / K +-ATPase, ATP5A (IM CVa), ubiquinone cytochrome c reductase core protein I (IM Core I), VDAC1 / porin (OM Porin), matrix cyclophilin D (Matrix CypD), intermembrane space cytochrome c (IMS Cytc), basement membrane-specific heparan sulfate proteoglycan core protein (HSPG2), plasma membrane calcium-transporting ATPase 1 (ATP2b1), endoplasmic reticulum membrane protein complex subunit 1 (EMC1), transmembrane protein 43 (TMEM43), vesicle membrane protein VIP36 (LMAN2), vesicle-associated membrane protein-associated protein A (VAPA), transmembrane 9 superfamily member 2 (TM9SF2), transmembrane emp24 domain-containing protein 10 (TMED10), adipocyte plasma membrane-associated protein (APAMP), synaptic vesicle membrane protein VAT (VAT1), nuclear pore membrane glycoprotein 210 (NUP210), plasma membrane calcium-transporting ATPase 4 (ATP2b4), plasma membrane calcium-transporting ATPase 2 (ATP2b2), erythrocyte band 7 integral membrane protein (STOM), transmembrane emp24 domain-containing protein 9 (TMED9), mitochondrial import inner membrane transposase subunit TIM44 (TIMM44), membrane-associated progesterone receptor component 1 (PGRMC1), ER membrane protein complex subunit 3 (EMC3), vesicle-associated membrane protein-associated protein B (VAPB), membrane primary amine oxidase (AOC3), vesicle-associated membrane protein 7 (VAMP7), Golgi membrane protein 4 (GOLIM4), membrane-associated progesterone receptor component 2 (PGRMC2), transmembrane 9 superfamily member 3 (TM9SF3), transmembrane 9 superfamily member 4 (TM9SF4), endoplasmic reticulum membrane protein complex subunit 2 (EMC2), mitochondrial import inner membrane transposase subunit TIM50 (TIMM50), peroxide Proteosomal membrane protein 11B (PEX11b), transmembrane emp24 domain-containing protein 2 (TMED2), secretory carrier-associated membrane protein 3 (SCAMP3), thioredoxin-related transmembrane protein 4 (TMX4), peroxisomal membrane protein PMP34 (SLC25a17), peroxisomal membrane protein PEX14 (PEX14), endoplasmic reticulum membrane protein complex subunit 8 (EMC8), interferon-induced transmembrane protein 3 (IFITM3), lysosomal-associated membrane glycoprotein 2 (LAMP2), thioredoxin-related transmembrane protein 2 (TMX2), vesicle-associated membrane protein 3 (VAMP3), lysosomal-associated membrane glycoprotein 1 (LAMP1), mitochondrial import inner membrane transposase subunit Tim8A (TIMM8a1), lysosomal membrane protein 2 (SCARB2), Iggamma-2A chain C region (IGHG2a), transmembrane 9 superfamily member 1 (TM9SF1), inner nuclear membrane protein Man1 (LEMD3), transmembrane emp24 domain-containing protein 4 (TMED4), Thy-1 membrane glycoprotein (Thy1), mitochondrial import inner membrane transposase subunit Tim23 (TIMM23), mitochondrial import inner membrane transposase subunit Tim9 (TIMM9), mitochondrial import inner membrane transposase subunit Tim13 (TIMM13), secretory carrier-associated membrane protein 2 (SCAMP2), secretory carrier-associated membrane protein 1 (SCAMP1), integral membrane protein 2B (ITM2b), mitochondrial import inner membrane transposase subunit Ti m10 (TIMM10), vacuole membrane protein 1 (VMP1), growth hormone-induced transmembrane protein (GHITM), death domain-containing membrane protein (NRADD), vesicle-associated membrane protein 8 (VAMP8), transmembrane anterior-posterior transition protein 1 (TAPT1), mitochondrial intermembrane space import and assembly protein 40 (CHCHD4), glycosylated lysosomal membrane protein (GLMP), transmembrane 4 domain superfamily A member 6D (MS4A6D), translocation chain-associated membrane protein 1 (TRAM1), interferon-induced transmembrane protein 2 (IFITM2), sarcolemma-associated protein (SLMAP), membrane magnesium transporter 1 (MMGT1), nuclear envelope pore protein POM121 (POM121), transmembrane protein C16orf54 homologous protein (AI467606), vacuolar ATPase assembly integral membrane protein Vma21 (VMA21), epithelial membrane protein 1 (EMP1), membrane-associated phosphatidylinositol transfer protein 1 (PITPNM1), small integral membrane protein 15 (SMIM15), nuclear integral membrane protein 1 (NEMP1), integral membrane protein GPR180 (GPR180), secretory carrier-associated membrane protein 4 (SCAMP4), translocation chain-associated membrane protein 2 (TRAM2), transmembrane and coiled-coil domain protein 3 (TMCC3), matrix membrane-associated protein 1 (SMAP1), neuronal membrane glycoprotein M6-b (GPM6b), epithelial membrane protein 2 (EMP2), Golgi membrane protein 1 (GOLM1), SI D1 transmembrane family member 2 (SIDT2), trans-Golgi network integral membrane protein 2 (TGOLN2), Golgi membrane protein TVP23 homolog B (FAM18b), mitochondrial inner membrane protein OXA1L (OXA1L), ossification-related transmembrane protein 1 (OSTM1), peroxisomal membrane protein PEX13 (PEX13), unilaterally passing membrane and coiled-coil domain protein 1 (SMCO1), distal membrane arm assembly complex protein 1 (DMAC1), secretory carrier-associated membrane protein 5 (Scamp5), cystic fibrosis transmembrane conductance regulator (CFTR), osteoclast-stimulating transmembrane protein (OCSTAMP), fat storage-inducing transmembrane protein 2 (FITM2) and transmembrane channel-like protein 5 (TMC5), and any combination thereof.

[0539] Transmission electron microscopy (TEM) images showed that all three membrane vesicle preparations (EM, TM, and HM) had similar sizes, approximately 100 nm in diameter, whereas the zeta potential of EM vesicles was more negative than that of TM and HM vesicles ( Figure 24 A-24C). The present invention evaluated the content of lipopolysaccharide (LPS) in all three membrane NPs (EM-NP, TM-NP and HM-NP) and found no significant difference between HM-NP and the LPS-free control group (p = 0.2977) ( Figure 25 ), indicating that most of the LPS was removed from the E. coli inner membrane during the preparation process. PLGA NPs were prepared using a double emulsion method. HM-NPs were obtained by repeatedly coextruding PLGA cores and HM vesicles through a liposome extruder with a 200 nm filter membrane pore size. Similarly, EM-coated PLGA NPs (EM-NPs) and TM-coated PLGA NPs (TM-NPs) were produced by repeatedly coextruding PLGA cores with EM vesicles or TM vesicles through a liposome extruder with a 200 nm filter membrane pore size. TEM images showed that HM-NPs had a uniform spherical nanostructure with an inner core and an outer shell ( Figure 29B and Figure 26 A-26B). More specifically, the diameters of the PLGA core and membrane shell of HM-NP were 86.83±7.41 nm and 14.16±2.07 nm, respectively ( Figure 26 A-26B). And when the mass ratio of nanoparticles to hybrid membrane is 5:1, the surface charge of HM-NPs is -21.3mV ( Figure 29C ), which is basically unchanged compared with the charge of HM vesicles ( Figure 24 C), indicating that at this ratio, the HM surface is intact and has reached saturation. Dynamic light scattering (DLS) analysis showed that the hydrated particle size of HM-NPs was approximately 180 nm ( Figure 29C ), which is similar to that of EM-NPs and TM-NPs. The difference in particle size between TEM and DLS results is attributed to the following reasons: TEM measures the diameter of the sample in the dry state, while DLS determines the hydrated particle size of the sample, taking into account the hydration layer on the particle surface. In the stability study, the size and zeta potential of HM-NPs were compared before freeze-drying and resuspended in phosphate-buffered saline (PBS; Figure 27 Furthermore, these physical properties of HM-NPs remained stable after they were suspended in PBS at 4°C for more than 3 weeks ( Figure 28 A-28B).

[0540] To identify the proteins contained in the membrane-coated NPs, the present invention resolved the proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained the resulting gel with Coomassie Brilliant Blue. The protein profile of HM-NPs was similar to that of the combination of proteins from two separate membrane NPs ( Figure 30 , EM-NP and TM-NP). To further characterize the properties of HM-NP, the present application examined the specific proteins of EM-NP, TM-NP and HM-NP ( Figure 29D FtsZ is a bacterial cell division protein that is present on both EM-NP and HM-NP ( Figure 29D ), while a series of mammalian cell proteins, including Na + / K + -ATPase, ATP5A (IM CVa), ubiquinol-cytochrome c reductase core protein I (IM Core I), VDAC1 / Porin (OM Porin), matrix cyclophilin D (Matrix CypD) and IMS cytochrome c (IMS Cytc) were all clearly present in TM-NP and HM-NP ( Figure 29D Furthermore, immunogold staining of the three membrane nanoparticles followed by TEM observation provided direct visual evidence for the presence of FtsZ and Na on HM-NPs, respectively.+ / K + -ATPase (specific markers for EM and TM, respectively) Figure 29E ).

[0541] Compared with the 10 nm gold particles in HM-NPs (with Na + / K + Compared with the 5 nm gold particles (bound to FtsZ, labeled EM) on the surface of HM-NPs, the abundance of 5 nm gold particles (bound to FtsZ, labeled EM) was greater ( Figures 31A-31B ), the corresponding fact is that the feed ratio of EM is larger than that of TM. In summary, the present application has successfully constructed hybrid membrane-coated nanoparticles that inherit key components from bacterial endomembranes and tumor cell membranes.

[0542] Example 13

[0543] HM-NPs enhance the uptake of tumor antigens, activate TLRs expression on the surface of BMDCs, and co-deliver antigens and adjuvants to BMDCs.

[0544] Internalization into DCs is the first step in the processing and presentation of tumor-associated antigens (TAAs). Therefore, this application studied the internalization of three membrane NPs by BMDCs. After incubating the membrane preparation loaded with rhodamine B with BMDCs for one hour, it was found that all three membrane nanoparticles labeled with rhodamine B were internalized into BMDCs and localized in lysosomal structures ( Figure 35A and Figure 32 Since most of the nanoparticles were captured into the lysosomal compartment, there was no statistical difference in the co-localization efficiency of the three membrane NPs with lysosomes ( Figure 33 We also used flow cytometry to study the ability of mouse BMDCs to take up each group of membrane nanoparticles. We synthesized a combination of NP (Mix NP, i.e., physically mixing EM-NP and TM-NP) or HM-NP with rhodamine B-loaded membrane samples and incubated them with BMDCs for 8 hours. We then evaluated the rhodamine B fluorescence intensity in each group of cells by flow cytometry ( Figure 35B ). The present application observed that BMDC took up only a small portion of TM-NPs (5.16%), which indicates that vaccines derived only from TM are not sufficient to effectively present antigens to APCs. In contrast, BMDC effectively took up two bacterial inner membrane-derived preparations. Samples containing EM (EM-NP, Mix NP and HM-NP) showed 20 times more cell-associated fluorescence than the TM-NP group. The reason for this is that DCs display a large number of PRRs on their surface, which helps to recognize EM surface PAMPs and helps to increase the uptake level of DCs. In addition, the absorption rate of HM-NPs is almost the same as that of EM-NPs ( Figure 35B). These results indicate that the introduction of bacterial inner membrane can promote the uptake of nanoparticles by BMDC, and that membrane fusion technology can give HM-NP the property of stimulating DC to take up tumor antigens. Studies have shown that TLRs can recognize bacterial surface PAMPs and initiate innate immune responses. In order to study the expression of TLRs on the surface of DCs stimulated by EM adjuvants, we co-incubated different groups of membrane nanoparticles (EM-NPs, TM-NPs, Mix NPs and HM-NPs) with BMDCs at 37°C for 24 hours. Subsequently, the whole cell surface protein of each group was extracted, and the protein expression level of each group was detected using a series of specific membrane surface TLRs protein antibodies TLR1, TLR2, TLR4 and TLR6. , the present application observed that the expression of TLR1, TLR2 and TLR6 in BMDCs in the HM-NP treatment group was higher than that in the control group ( Figure 35C ), proving that HM-NPs can stimulate the expression of multiple membrane TLRs on the surface of APCs, showing good potential for innate immune stimulation. This application found that there was no significant difference in TLR4 expression among all groups, which may be due to the removal of most LPS during the preparation of HM-NPs, resulting in no significant change in the expression of its downstream TLR4 receptors ( Figure 25 ). TLR stimulation regulates the production of pro-inflammatory cytokines through downstream pro-inflammatory signaling. Compared with the control group, the present application observed that the expression of NF-κB molecules in the HM-NPs group was significantly upregulated ( Figure 35C ). Figure 35D Possible known signal transduction mechanisms are shown in .

[0545] In order to evaluate the innate immune stimulation level of EM on BMDCs, this application first examined the ability and changes of EM-NPs alone to stimulate the secretion of proinflammatory cytokines after incubation with BMDCs for a certain period of time. We selected a series of EM-NPs with a concentration gradient (0.12 mg / mL, 0.25 mg / mL, 0.50 mg / mL, 1.00 mg / mL) to incubate with BMDCs, and selected different time points (0h, 3h, 8h, 24h, 48h) to collect the supernatant of each group of cells, and used a kit to detect the secretion level of the proinflammatory cytokine IL-6 in the supernatant. It was observed that the secretion level of IL-6 showed a clear concentration and time dependence, indicating that bacterial-derived membrane vesicles can stimulate BMDC activation and activate the innate immune response ( Figure 34 To further investigate the ability of different groups of membrane nanoparticles to stimulate BMDCs to secrete proinflammatory cytokines, different membrane NPs were co-incubated with BMDCs for 24 h, and then the expression of specific cytokines ( Figures 35E-35G). Compared with the TM-NP group, the present application observed a large release of IL-6, TNF-α, and IL-1β in BMDCs treated with HM-NPs. This striking cytokine release profile in HM-NPs is attributed to the adjuvant properties of the bacterial cytoplasmic membrane components, as TM-NPs lack the ability to stimulate BMDCs. In addition, HM-NPs are more effective than Mix NPs in stimulating IL-6 and IL-1β secretion (p < 0.0001). Compared to the mixed delivery of individual components, tumor autologous antigens and immune adjuvants on the same nanoparticle have the potential to enhance the intensity of immune stimulation.

[0546] Next, the present application investigated the ability of HM-NPs to promote BMDC maturation. BMDCs were treated with different samples for 24 hours, and then the cells were stained with flow cytometry antibodies CD11c (a marker of DC), CD80 (a stimulatory marker of mature DC), and CD86 (a stimulatory marker of mature DC). The fluorescence intensity of the cells was then measured by flow cytometry ( Figures 35H-35I ). This is consistent with the results of BMDC activation ( Figures 35E-35G ), the percentage of mature BMDCs in all EM-containing nanoparticles (EM-NP, Mix NP and HM-NP) was significantly increased (p < 0.0001). As indicated by the enhanced expression of CD80 and CD86. The present application also observed that BMDCs failed to achieve efficient maturation when incubated with TM-coated nanoparticles ( Figures 35H-35I These results indicate that the bacterial inner membrane confers the ability of HM-NPs to promote DC maturation. Again, similar to the DC activation results ( Figures 35E-35G ), the proportion of mature BMDCs treated with HM-NPs was greater than that in the Mix NP group (p<0.0001), further confirming the advantage of HM-NPs in enhancing innate immune response ( Figures 35H-35I ). It is worth noting that there was no significant difference in BMDC internalization rate between HM-NP and Mix NP (p>0.9999), but there was a significant difference in their innate immune stimulation ability ( Figure 35B To explain this phenomenon, we hypothesize that, compared with the physical mixing of separate antigens and adjuvants, the nanosystem wrapped in the same membrane can achieve a higher level of antigen-adjuvant co-delivery, resulting in a stronger synergistic immune effect.

[0547] To verify the above hypothesis, the colocalization efficiency of EM and TM components in BMDCs treated with HM-NPs and Mix NPs was further investigated using immunofluorescence experiments. BMDCs were incubated with HM-NPs or Mix NPs for 8 h. Red fluorescent-labeled FtsZ antibodies and green fluorescent-labeled Na + / K +-ATPase antibodies were used to identify EM and TM ( Figure 35J Immunofluorescence images showed that when BMDCs were treated with HM-NPs, the colocalization was significantly greater than that in the Mix NP group (p < 0.0001) (average 81.53% vs 52.39%; Figure 35K These results demonstrate that HM-NPs have an enhanced ability to deliver EM and TM to the same DCs compared to mixed samples. Co-delivery of antigens and adjuvants to DCs can enhance the immunogenicity of tumor antigens and the efficacy of cancer vaccines in immunotherapy. The results of this application validate that HM-NPs can facilitate this co-presentation.

[0548] Example 14

[0549] Detection of HM-NPs promoting DC maturation in LNs and activating tumor membrane antigen-specific T cells

[0550] To assess DC maturation in vivo, female BALB / c mice were subcutaneously inoculated with murine 4T1 breast cancer cells. 3 The majority of the tumor tissue from each mouse was removed to prepare the vaccine formulation, with the remaining approximately 1% used to simulate the presence of residual microtumors in the surgical bed of the clinic. The mice were imaged and the tumors were measured on the second day after surgery and randomly divided into five groups. The mice were immunized with different vaccine formulations on days 3, 5, and 9 after surgery. 12 hours after the last vaccination, the present application evaluated the presence of stimulatory molecules on DCs in the inguinal lymph nodes. Figures 37A-37B As shown in Figure 3, the expression of two stimulatory markers (CD80 and CD86) increased significantly after DC stimulation. Mice treated with all three membrane-coated nanoparticles (including TM-NP) showed the highest expression in the HM-NP group (expression of CD80 and CD86 in the HM-NP, EM-NP and Mix groups was 14.38%, 12.36% and 13.82%; respectively, 14.46%, 9.76% and 11.92%). Figures 35H-35I ) compared to the efficacy of TM-NPs in vivo ( Figures 37A-37B ), which may be because after TM-NPs enter the mouse body, they are recognized by sensitized immune cells (such as DCs) in the immune system as pathogen damage-associated molecular patterns (DAMPs), which stimulates the maturation of DCs to a certain extent. The HM-NP treatment group showed the highest expression of co-stimulatory markers, which is consistent with the in vitro maturation results ( Figures 35H-35I ).

[0551] To present tumor membrane-associated antigens to naive T cells via activated DCs, the antigens must migrate to lymph nodes (LNs). To determine whether HM-NPs can accumulate in LNs, we subcutaneously injected membrane NPs labeled with the fluorescent dye IR-780 into BALB / c mice. 12 hours later, inguinal LNs were excised and imaged using an in vivo imaging system (IVIS; Figure 36A ) imaging. Fluorescence signals were observed in all groups, and the fluorescence intensity of the HM-NP group was 3.3 times that of the EM-NP group and 12.4 times that of the TM-NP group ( Figure 36B ), indicating that HM-NPs most effectively migrated to LNs for tumor antigen presentation.

[0552] To further verify whether HM-NP can effectively activate naive T cells, the present invention subcutaneously inoculated female BALB / c mice with murine 4T1 breast cancer cells. The surgical procedure and vaccination procedures were performed as described above. To evaluate the antigen-specific CD8 + T cells, spleen cells were collected 12 hours after the final vaccination and incubated with 4T1 cell membranes (as antigens) for 24 hours. The results showed that almost no positive spots were observed in the TM-NP treatment group, indicating that the tumor membrane preparation alone had limited activation of T cells ( Figures 37C-37D ). At the same time, a moderate number of T cells were activated in the EM-NP treatment group. This weak immune response may be due to the lack of tumor membrane antigens. The Mix NP and HM-NP groups, which contain both antigens and adjuvants, showed more positive spots than the other groups. These results reflect the weak immunogenicity of the tumor membrane, which can be enhanced by the presence of EMs. The co-delivery of antigens and the adjuvant strategy caused an approximately two-fold increase in IFN-γ release by T cells compared with the Mix NP treatment group. In addition, the production of tumor membrane antigen-specific cytokines in T cells, assessed by flow cytometry, confirmed that HM-NPs had a much stronger ability to enhance specific T cell responses than other preparations ( Figures 37E-37F These results indicate that HM-NPs can achieve DC maturation and splenic T cell activation through the co-delivery of tumor antigens and adjuvants.

[0553] Detection of low systemic inflammatory response induced by HM-NPs

[0554] Enhanced stimulation of the patient's immune system during cancer immunotherapy, as well as the integration of bacterial membrane components in the formulation, can lead to life-threatening side effects such as cytokine storms. To evaluate the intensity of the systemic inflammatory response stimulated by the vaccine formulation, female BALB / c mice were subcutaneously inoculated with murine 4T1 breast cancer cells. The surgical and vaccination procedures were performed as described above. The concentrations of inflammatory cytokines and chemokines in serum were measured by Luminex magnetic bead-based ELISA kits in the ProcartaPlex multiplex immunoassay. Overall, serum concentrations of IL-6, IFN-γ, TNF-α, MCP-1, IL-12p70, IL-1β, IL-23, IL-27, and IL-17A were increased in patients treated with HM-NPs ( Figure 37G and Figure 38 A-38I). However, only two proinflammatory cytokines, IL-6 and IL-1β, were increased in HM-NP-treated mice compared with control mice (p = 0.0269; p = 0.0139). The remaining 11 cytokines or chemokines were expressed similarly in all groups, indicating that the inflammatory response induced by HM-NPs was controllable ( Figure 37G and Figure 38 A-38I). Taken together, HM-NPs can enhance both innate and adaptive immunity and stimulate low systemic inflammatory responses, indicating that this formulation has the potential to safely elicit anti-tumor immunotherapy efficacy.

[0555] Example 15

[0556] Detection of HM-NP vaccination-induced tumor regression in the murine 4T1-Luc tumor model

[0557] After confirming that HM-NP can activate DCs and T cells in vivo, this application set out to evaluate the ability of HM-NP vaccine to inhibit tumor recurrence ( Figure 39A To this end, the present application followed the surgical and vaccination procedures, subcutaneously injected female BALB / c mice with mouse 4T1-Luc breast cancer cells, and monitored the subcutaneous tumors of the mice using IVIS before and after surgery ( Figure 39B IVIS images showed that tumor volume gradually increased in all non-HM-NP vaccination groups. Figures 39C-39E Tumor growth was not affected by EM-NP or TM-NP vaccination. Although the Mix NPs formulation delayed tumor recurrence, the antitumor effect was still mild, with a survival rate of 25% within 60 days ( Figure 39E, n = 12). In contrast, mice treated with the HM-NP vaccine demonstrated enhanced tumor suppression, with a survival rate of approximately 92%, further confirming the benefits of using the hybrid membrane nanoplatform to co-deliver tumor antigens and adjuvants. Importantly, no tumor recurrence was observed in mice that survived to the experimental endpoint (day 60), indicating a significantly higher survival rate than in other groups.

[0558] Example 16

[0559] HM-NP vaccine can inhibit tumor regression in multiple murine tumor models

[0560] To demonstrate the universality of HM-NP in postoperative immunotherapy, female BALB / c mice were subcutaneously inoculated with CT26 colon tumor cells. Figure 40A ) was the same as above. Tumor volume was measured with an electronic caliper and the survival of the three different groups was monitored over 60 days. Figures 40B-40D As shown in Figure 2, in the HM-NP group, vaccination after surgical treatment effectively inhibited the recurrence of CT26 tumors at the surgical site. Similar to the results of the 4T1-Luc model described above ( Figures 39A-39E ), the Mix NP formulation delayed tumor recurrence by only eight days compared to the untreated group. No mice in the Mix NP group survived to the 60-day time point. Mice vaccinated with HM-NP showed no tumor recurrence for 60 days, while the Mix NP group had a median survival of approximately 37 days ( Figure 40E ). It is worth noting that in this experiment, all mice immunized with HM-NPs achieved effective tumor recurrence inhibition, with an inhibition rate of up to 100%.

[0561] The CT26 and 4T1-luc models have higher immunogenicity and are more responsive to immunotherapy. To further demonstrate the therapeutic potential of the HM-based vaccine of this application, this application tested the formulation in two tumor models with lower immunogenicity, the B16F10 melanoma and EMT-6 breast tumor models. Similar to the results of this application in the CT26 and 4T1-Luc models, more than 90% of the mice vaccinated with HM-NP had no tumor recurrence within 60 days, while 40%-100% tumor recurrence was observed in the Mix NP and control groups. B16F10 melanoma ( Figure 40F ) and EMT-6 breast tumors ( Figure 40G Similarly, the results of this application show that vaccines based on the hybrid membrane strategy can be applied to a variety of solid tumor models with significant therapeutic efficacy.

[0562] The present application also compared the effects of bacterial endomembranes used as vaccine adjuvants with the commercial adjuvant monophosphoryl lipid A (MPLA) in a mouse CT-26 tumor model ( Figures 41A-41B ). Meanwhile, the present application also investigated the anti-tumor efficacy of hybrid membrane vesicles without a PLGA core. EM-based cancer vaccine formulations consistently demonstrated higher therapeutic efficacy than other tumor membrane antigen-containing formulations, including HM vesicles and three MPLA-containing groups (TM-NP+free MPLA, TM-MPLA-NP, and TM-NP). Figures 41A-41B The present application observed that the complete remission rate (CR) was 100% in the HM-NP group, 62.5% in the HM vesicle group, and 50.0%-62.5% in the three MPLA-containing groups ( Figures 41A-41B ), the results showed that the vaccine formulation with hybrid membrane-coated PLGA NPs was the best formulation among the experimental groups.

[0563] Example 17

[0564] HM-NP vaccination protects against tumor remodeling in the CT26 tumor model

[0565] This study further investigated whether HM-NPs could provide long-term protection for more than 60 days in the CT-26 tumor rechallenge model. In this model, all mice from the HM-NPs vaccination group of the CT-26 tumor model were randomly divided into three groups and inoculated with saline, CT-26 colon adenocarcinoma cells, or 4T1 breast cancer cells. Figures 42A-42B As shown, mice inoculated with CT-26 tumor cells showed complete tumor elimination and 100% tumor inhibition rate. In addition, mice in the saline group did not experience tumor recurrence for 90 days. However, the tumor volume of mice inoculated with 4T1 cells reached 1000 cm after 3 weeks. 3 , indicating that the protective effect provided by HM-NP is a CT-26-specific immune response. In addition, the present application found that the serum concentrations of proinflammatory cytokines (including IL-6, IL-1β and TNF-α) in mice vaccinated with CT-26 cells increased compared with the 4T1-vaccinated group. CT26 tumor re-challenge model ( Figures 42C-42E In addition, the secretion of the specific immune cytokine IFN-γ was also increased in the CT-26 vaccination group ( Figure 42F Among T cell subsets, HM-NPs also increased effector memory T cells compared to naive T cells ( Figures 42G-42H These results are consistent with a stronger immune response due to the presence of the same tumor antigens. Therefore, the HM-NP vaccine not only provides immunity against tumor recurrence but also provides specific long-lasting protection.

[0566] Analysis of innate and adaptive immunity is required for tumor suppression after HM-NP vaccination

[0567] NK cells and macrophages play an important role in innate immunity, and CD4 + T cells and CD8 + T cells are crucial for adaptive immune responses. To explore the potential mechanism by which HM-NPs induce enhanced anti-tumor responses, mice were inoculated with CT-26 murine colon adenocarcinoma cells and the tumors were excised as described above. Female BALB / c mice were subcutaneously inoculated with CT-26 cells. When the tumor volume reached approximately 300 mm, the tumor was resected and the tumor was resected. 3 Then, the mice were randomly divided into six different groups. Antibodies targeting immune cell surface markers were injected intraperitoneally (Table 3). At the beginning of HM-NP treatment, macrophages (CSF1R antibody), NK cells (ASGM1 antibody), CD8 + T cells (CD8 antibodies) or CD4 + T cells (CD4 antibody) one day before. Peripheral blood mononuclear cells (PBMC; Figure 43 Flow cytometry of the HM-NPs and saline (as a control) was performed to confirm the depletion of specific cell types. Mice were then inoculated with HM-NPs or saline (as a control) three times on days 3, 5, and 9 after surgery. Figure 42I and Figure 44A As shown, mice vaccinated with HM-NPs achieved 100% tumor remission when their immune systems remained intact. However, despite vaccination with HM-NPs, all CD8-depleted mice still experienced tumor recurrence, revealing that CD8 + The major role of T cells in tumor regression ( Figure 42I and Figure 44B NK cell depletion also resulted in a significant decrease in overall survival, whereas CD4 + T cell blockade did not significantly affect (p = 0.3173) the efficacy of HM-NP vaccination ( Figure 44B Macrophage restriction resulted in a degree of tumor recurrence in HM-NP-inoculated mice, with tumor recurrence rates as high as 40% ( Figure 42I and Figure 44B In summary, antibody depletion studies have shown that CD8 + Both T cells and NK cells are crucial for tumor suppression, and other immune cell subsets also play a role in tumor regression.

[0568] Table 3 Selective cell type depletion during HM-NP treatment

[0569]

[0570] The peripheral blood of mice was analyzed by flow cytometry to confirm the expression of macrophages (CSF1R antibody), NK cells (ASGM1 antibody), CD8 +T cells (CD8 antibodies) or CD4 + Depletion of T cells (CD4 antibodies) Figure 43 ).

[0571] Example 18

[0572] Safety testing of HM-NP vaccine

[0573] Since bacterial components are present in the vaccine formulation, the biosafety of the vaccine was evaluated. Many bacteria are pathogenic due to the production of hemolysins. Therefore, a hemolysis assay was performed in which red blood cells were treated with a range of HM-NP concentrations. No significant hemolysis was observed with any of the HM-NP concentrations tested ( Figure 45 Next, at the end of the tumor regression experiment in the 4T1-Luc model, the present application harvested and processed the main organs of the mice in each group for hematoxylin-eosin (H&E) staining. No obvious histological changes were observed in any organs of the mice examined ( Figure 46 ). In addition, studies have shown that pathogen-derived molecules in bacterial membranes can cause liver and kidney damage through various causes, so liver and kidney function were evaluated in the mouse groups. There was no statistical difference in serum concentrations of liver enzymes (ALT and AST) in the HM-NP-treated mice ( Figure 47 A-47B). Serum concentrations of renal function markers (BUN and CREA) were approximately the same in all groups ( Figure 47 C-47D). Taken together, these data suggest that HM-NP has good biosafety and can be used as a highly effective and safe postoperative therapeutic vaccine for patients with solid tumors, with the potential for clinical translation.

[0574] Example 19

[0575] Differential analysis of bacterial outer membrane vesicles (OMVs) and bacterial inner membranes

[0576] OMV proteomic analysis:

[0577] (1) Extracting Escherichia coli outer membrane vesicles and freezing them;

[0578] (2) Protein concentration was determined using the BCA method;

[0579] (3) Filter Aided Proteome Preparation (FASP) enzymatic hydrolysis;

[0580] (4) The peptides were concentrated and dried by centrifugation, desalted on a ZipTip C18 column, and dried before being analyzed by mass spectrometry;

[0581] (5) Add 30 μL of loading buffer (acetonitrile: water: formic acid = 2:98:0.1) to the dried sample, shake to dissolve, transfer to a sample bottle, and analyze by mass spectrometry.

[0582] Database search and results: This experiment adopts the basic process of proteomic identification based on mass spectrometry, that is, the MS / MS mass spectrometry data is subjected to a series of optimization processes and then compared with the database for similarity scoring to perform protein identification. Each raw data file and the corresponding database are uploaded to Maxquant 1.5.8.3 software respectively. After the database search is completed, the Reverse and Potential Contaminant proteins are removed, and the database search results are statistically analyzed: This mass spectrometry search detected a total of 218 total proteins (non-redundant), 441 total peptides (include redundant), 430 specific peptides (razor+unique), and 1230 total spectra (MS / MS Identified). This application statistically analyzed the cellular location of proteins and found that proteins located in the cytosol accounted for the largest proportion, as high as 34.63%. The specific results are shown in Tables 4 and 5:

[0583] Table 4 Distribution of proteins in E. coli outer membrane vesicles

[0584] protein ％ cytoplasm 34.63 Cytoplasmic large ribosomal subunit 10.51 cytoplasm 10.12 outer membrane boundary periplasmic space 7.39 Cytoplasmic small ribosomal subunit 7.00 membrane 6.23 plasma membrane 5.06 periplasmic space 3.89 cell membrane 3.89 Components of the membrane 3.50 Components of the plasma membrane 1.56 Components of the cell outer membrane 1.17 pore complex 1.17 GroEL-GroES complex 0.78 Exodeoxyribonuclease VII complex 0.78 outer membrane 0.78 Nucleosides 0.78 ATP-binding cassette (ABC) transporter complex 0.78

[0585] In addition, this application also selected 10 representative key proteins and described their molecular weight and function. The results are shown in the following table:

[0586] Table 5 Expression of major proteins in E. coli outer membrane vesicles

[0587]

[0588] Functional Description:

[0589] OmpA: Outer membrane protein A, commonly found on the surface of Gram-negative bacteria, has a β-barrel structure. Functionally, it provides permeability to the outer membrane and maintains the stability of the outer membrane structure. It plays a vital role in bacterial life activities: it can act as an adhesin and invadin for bacteria, participate in the formation of biofilms, and also serve as a receptor for a variety of bacteriophages, making it an important target of the innate immune system.

[0590] OmpC: Outer membrane protein C, a pore protein in the outer membrane of Gram-negative bacteria, allows small molecules to diffuse passively across the outer membrane.

[0591] OmpX, or outer membrane protein X, is an important bacterial outer membrane protein belonging to the Ail protein family. In other bacteria, this protein participates in bacterial adhesion and invasion of cells, counteracting host immune defenses, and is closely associated with bacterial virulence. Therefore, it is considered a potential candidate target for vaccine development.

[0592] OsmE: Osmotic-induced lipoprotein E, which is related to osmotic pressure response;

[0593] LamB: The maltoporin is involved in the transport of maltose and maltodextrins and is essential for the transport of dextrins containing more than three glucosyl moieties. A hydrophobic path of aromatic residues ("lipidic") guides and selects sugars for transport through the channel. It also serves as a receptor for several bacteriophages, including lambda.

[0594] MipA: MltA-interacting protein, which acts as a scaffold protein required for the formation of a complex with MrcB / PonB and MltA. This complex may play a role in the expansion and separation of mural protein vesicles.

[0595] Lpp: The outer membrane lipoprotein Lpp interacts covalently and non-covalently with peptidoglycan. This interaction helps maintain the structural and functional integrity of the cell envelope.

[0596] SlyB: outer membrane lipoprotein SlyB, which is directly regulated by the PhoP gene, which is mainly involved in regulating the expression of bacterial virulence genes;

[0597] OmpW: Outer membrane protein W, a pore protein of the outer membrane of Gram-negative bacteria, is a receptor for colicin S4 and can be used as a target for drug screening;

[0598] FhuE: FhuE receptor is a receptor protein on the outer membrane of Gram-negative bacteria. It absorbs Fe3 through the co-biotics, ferrous amine B and azalea acid. + Necessary receptor molecules.

[0599] Proteomic analysis of the inner membrane of Escherichia coli:

[0600] (1) Four batches of Escherichia coli inner membranes were extracted and frozen for storage;

[0601] (2) Protein concentration was determined using the BCA method;

[0602] (3) reductive alkylation;

[0603] (4) Filter Aided Proteome Preparation (FASP) enzymatic hydrolysis;

[0604] (5) The peptides were concentrated and dried by centrifugation, desalted on a ZipTip C18 column, and dried before being analyzed by mass spectrometry;

[0605] (6) The peptides were concentrated and dried by centrifugation, desalted on a ZipTip C18 column, and then dried and prepared for mass spectrometry analysis.

[0606] Database search and results: This experiment adopts the basic process of proteome identification based on mass spectrometry, that is, after a series of optimization processing of MS / MS mass spectrometry data, similarity comparison and scoring are performed with the database to identify proteins. We use Proteome Discoverer 2.4 to search the database. After the search, the Reverse and PotentialContaminant proteins are removed, and the search results are counted: This mass spectrometry search detected a total of 2302 total proteins (non-redundant), 20454 total peptides (include redundant), 19006 specific peptides (razor+unique), and 86026 total spectra (MS / MS Identified). This application counted the cellular locations of proteins and found that proteins located in the intracellular (intracellular) accounted for the largest proportion. The specific results are as follows Figure 48 As shown in Table 6:

[0607] In addition, this application also selected 12 representative proteins and described their protein scores, molecular weights, and functions. The results are shown in the following table:

[0608] Table 6 Expression of main proteins of the inner membrane of E. coli in different batches

[0609] Protein name Protein accession number Protein database scoring Molecular weight [KDa] type FqV P0A9A6 991 40.3 cell division proteins Yj Y P0ADW3 125 15.0 Inner membrane proteins YidC P25714 109 61.5 inner membrane protein translocase MsbA P60752 69 64.4 ABC transporters TatA P69428 58 9.7 Inner membrane transport proteins MlaA P76506 43 28.0 Intermembrane phospholipid transport system lipoprotein TolQ P0ABU9 26 25.6 Inner membrane proteins YebE P33218 24 23.7 Inner membrane proteins SecB C4ZXK1 63 17.3 Protein exporters FqV P10121 103 54.5 signal recognition granulin receptor Ffh P0AGD8 174 49.8 signal recognition granule protein YajC P0ADZ8 70 11.9 Sec translocon accessory complex subunit

[0610] Functional Description:

[0611] FtsZ, also known as cell division GTPase, is a cell division protein and a well-characterized protein in the bacterial cell division apparatus. It accumulates during the early to mid stages of bacterial cell division and plays a crucial role in septum formation in most bacteria. It has also been proposed to be the bacterial cytoskeletal counterpart of eukaryotic microtubules.

[0612] YhcB: a bacterial inner membrane protein involved in cytoskeleton and peptidoglycan biosynthesis;

[0613] YidC: an inner membrane protein translocase that is related to the Sec translocon and is an auxiliary factor for the integration, folding and assembly of inner membrane proteins. It can transport small inner membrane proteins (especially inner membrane proteins with small periplasmic domains) into the inner membrane.

[0614] MsbA: an ABC transporter and a lipid A export ATP binding / permease protein;

[0615] TatA: An inner membrane transport protein and a Sec-independent protein translocase protein, it is a member of the Tat family. Tat is a system in Escherichia coli that can transport folded proteins across the membrane, and its signal peptide contains a highly conserved twin-arginine motif. The Tat family members include four proteins: TatA, TatB, TatC, and TatE. Their complex forms a transport channel on the plasma membrane of Escherichia coli. The substrate proteins transported by the Escherichia coli Tat system are mostly components of the respiratory electron transport chain and are related to many life activities of Escherichia coli.

[0616] MlaA: A lipoprotein in the membrane phospholipid transport system, responsible for the transport and exchange of phospholipids between the inner and outer membranes of bacteria;

[0617] TolQ: An inner membrane protein that belongs to the Tol-Pal system. The Tol-pal inner membrane system is one of the inner membrane protein systems of Gram-negative bacteria. The name of the Tol protein in the system is related to resistance to colicin. Tol-pal is composed of five proteins: TolQ, TolA, TolB, TolR and Pal. Their genes are located on the same operon. TolB is a periplasmic protein, and proteins TolA, Q, and R are three inner membrane proteins that interact with each other to form an inner membrane protein complex. The TolQ protein is related to bacterial cell division;

[0618] YebE: an inner membrane protein and a heat shock response molecular tag of Escherichia coli;

[0619] SecB, a protein exporter. The signal recognition particle (SRP) targets inner membrane proteins to the inner membrane co-translationally, whereas the SecB pathway targets secretory proteins in a later co-translational or post-translational process;

[0620] FtsY: A signal recognition particle protein receptor. The Escherichia coli SRP receptor contains only one subunit, FtsY, a homolog of mammalian SRα. E. coli FtsY is evenly distributed between the cytoplasm and the inner membrane. FtsY binding to the membrane involves lipids and a protein membrane factor, most likely the Sec-translocon component SecY. Interaction with phospholipids stimulates FtsY GTPase activity.

[0621] Ffh: A signal recognition particle protein homologous to mammalian SRP54. Ffh is essential for the growth of Escherichia coli. It has been proposed that SRP54 (Ffh) in its activated, GTP-bound form, bound to the ribosomal targeting signal, can be used for interaction with SR (FtsY). The GTPases of Ffh and FtsY stimulate each other upon complex formation and are proposed to function as GTPase activators.

[0622] YajC: a Sec translocon accessory complex subunit involved in bacterial inner membrane protein biogenesis.

[0623] Example 20

[0624] The ability of five bacterial species, including Escherichia (Gram-negative), Staphylococcus (Gram-positive), Bacillus (Gram-positive), Lactobacillus (Gram-positive), and Pseudomonas (Gram-negative), to inhibit tumor recurrence after surgery when combined with cell membranes derived from autologous tumor tissue.

[0625] In this example, HM-NPs were prepared by coating PLGA with hybrid membranes composed of five different bacterial endomembranes and tumor cell membranes. These included Escherichia, Staphylococcus, Bacillus, Lactobacillus, and Pseudomonas. The total membrane protein concentrations of the five products were kept consistent. A tumor membrane alone group and a saline solution group were also included as controls. The ability of the different bacterial endomembranes to inhibit postoperative tumor recurrence was demonstrated using a mouse colon cancer model. The specific steps are as follows:

[0626] (1) Extraction of the inner membrane of different bacteria: The specific operation is as follows: amplify the bacteria and culture them in culture medium to an OD600 value of 1.2; add lysozyme (to lyse the cell wall) to a final concentration of 2 mg / mL and incubate with the bacteria at 37°C for 2-3 hours; centrifuge and remove the supernatant to obtain protoplasts; add extraction buffer and use density gradient centrifugation to obtain the bacterial inner membrane; resuspend and store at -80°C until use;

[0627] (2) Female BALB / c mice were randomly divided into 7 groups, with 8-10 mice in each group. 200,000 CT26 colon cancer cells were subcutaneously inoculated on the right back of each mouse to establish a mouse colon cancer model (day 0);

[0628] (3) Observe and record the tumor growth of mice every other day. When the average tumor volume reaches 300 mm 3 Around 24 h, all mouse tumors were surgically removed, and the wounds were sutured (day 7);

[0629] (4) Extract the cell membrane of tumor cells from CT26 colorectal cancer tissue. The tumor tissue obtained in (3) was initially minced with scissors, and then a solution containing collagenase IV (1.0 mg mL -1 ), DNase (0.1 mg mL -1 ) and hyaluronidase (0.1 mg mL -1 ) GBSS solution at 37°C for 15 minutes to digest the tumor tissue; the digested tissue was ground, then filtered through a 0.22 μm pore size filter, and the filtrate was centrifuged; the centrifuged cells were resuspended, and an extraction buffer containing mannitol, sucrose, and EGTA was added. The cells were then disrupted using a cell disruptor in an ice bath, and finally, tumor cell membranes derived from the tumor tissue were obtained by centrifugation; the cell membranes were resuspended and frozen at -80°C until use;

[0630] (5) 3 mg of different bacterial endomembranes were mixed with 1 mg of extracted surgically removed tumor cell membranes in PBS and shaken at 37°C for 15 min. The mixed membrane solution was then passed through a liposome extruder with a filter membrane pore size of 400 nm for a total of 13 times (one back and forth counted as one time) to obtain HM hybrid membrane microparticles;

[0631] (6) Dissolve 10 mg of PLGA in 1 mL of dichloromethane, and then add 0.2 mL of sterile distilled water; the mixture is ultrasonically emulsified on ice at 25W for 3 minutes; then mix with 2 mL of 1% sodium cholate solution (surfactant) and ultrasonically emulsify on ice at 30W for 5 minutes; add the emulsion dropwise to 10 mL of 0.5% sodium cholate solution, stir at 25°C for 15 minutes; rotary evaporate at 37°C for 10 minutes; centrifuge the emulsion at 10,000 g for 15 minutes; wash twice with sterile distilled water, and resuspend to obtain a PLGA polymer solution;

[0632] (7) 50 μL of 2 mg / mL HM hybrid membrane microparticles prepared in step (5) was mixed with 500 μL of 1 mg / mL PLGA prepared in step (6) and passed through a liposome extruder with a filter membrane pore size of 200 nm. The mixture was extruded back and forth 13 times (one back and forth counted as one time) to obtain HM-NPs hybrid membrane nanoparticles.

[0633] (8) On days 10, 12, and 16, mice in each group were subcutaneously injected with SM-NPs, TM-NPs, Mix NPs, and HM-NPs (100 μg / mouse). The saline group served as the control group.

[0634] (9) Observe and record the tumor growth of mice every other day. When the tumor volume of mice reaches 1000mm 3 The mice were considered dead when the CT scan was taken, and the specific time of death of each group of mice was recorded.

[0635] The results show that the HM-NPs hybrid membrane nanoparticles prepared from different bacterial inner membranes in this application have uniform particle size ( Figure 49A ), inhibiting the potential of tumor recurrence in mice after colon cancer surgery, and the inhibition rate was significantly improved ( Figure 49B ).

[0636] Example 21

[0637] This application is used for tumor inhibition experiments of liver cancer, gastric cancer, kidney cancer, pancreatic cancer, ovarian cancer, lymphoma, osteosarcoma, glioma, prostate cancer and melanoma.

[0638] The experimental process is as follows Figure 50A As shown:

[0639] (1) 6-8 week old C57BL / 6J or Balb / C mice were randomly divided into two groups, with 8-10 mice in each group. Different tumor cells were subcutaneously inoculated on the right back of each mouse. The inoculated tumor cell types and numbers, as well as the corresponding mouse strains, are shown in Table 7. Different mouse models of postoperative tumor recurrence were constructed to evaluate the inhibitory effect of the present invention on various tumors.

[0640] (2) When the average tumor volume reaches 300 mm 3 Around 24 hours after surgery, 99% of the tumor was removed, with 1% of the tumor remaining, and the wound was sutured to simulate postoperative tumor recurrence (day 0);

[0641] (3) Grind and crush the cut tumor mass, digest the tumor tissue with a mixed solution of collagenase, DNAse and hyaluronidase at 37°C, filter the digested cell suspension and then centrifuge, discard the supernatant after centrifugation, add extraction buffer and resuspend to obtain a cell suspension; use a cell disruptor to perform ice bath ultrasonic crushing on the cell suspension (35W, 5min); centrifuge the crushed cell suspension (3000g, 5min, 4°C); take the supernatant from the previous step and centrifuge again (10000g, 10min, 4°C); take the cell supernatant after centrifugation and add it to an ultracentrifuge tube for ultracentrifugation (100000g, 2h, 4°C); discard the supernatant after ultracentrifugation, resuspend with 1mL PBS to obtain tumor cell membrane fragments TM derived from surgical resection, and store at -80°C;

[0642] (5) Prepare various membrane nanoparticles according to the methods in the examples of this application, take electron microscope images, and characterize the hybrid membrane nanoparticles prepared from different tumor cells;

[0643] (6) Mice were immunized three times with HM-NPs (100 μg / mouse) prepared using the corresponding tumor membrane on days 3, 5, and 9 after surgery, and the normal saline group served as the control group (Control);

[0644] (7) Observe and record the growth of mouse tumors. When the volume of mouse tumors reaches 1000 mm 3 The mice were considered dead when the number of mice died, and the specific time of death of each group of mice was recorded. A survival curve was drawn.

[0645] Table 7 Cells and mice used in the construction of different tumor models

[0646]

[0647] like Figure 50B As shown, different tumor cells can be prepared with the Escherichia coli inner membrane into uniform size and possess the characteristics of core-shell hybrid membrane nanoparticles, proving that the present application can be used to prepare hybrid membrane nanoparticles containing liver cancer cells, gastric cancer cells, kidney cancer cells, pancreatic cancer cells, ovarian cancer cells, lymphoma cells, osteosarcoma cells, glioma cells, prostate cancer cells and melanoma cells.

[0648] like Figure 50C As shown in the figure, in the corresponding tumor models, the hybrid membrane nanoparticle vaccine (HM-NPs) significantly prolonged the survival of different tumor-bearing mice compared with the control group (Control) without any treatment.

[0649] Example 22

[0650] The present invention uses a hybrid membrane to wrap PLGA polymer. After passing through a liposome extruder with a pore size of 200 nm, the surface potential of the hybrid membrane shell can reach -50 mV. After adding cationic liposomes to the hybrid membrane, the surface potential of the hybrid membrane shell can reach +50 mV. The results are as follows: Figure 51 shown.

[0651] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed in the present application are obvious to those skilled in the art and are intended to be within the scope of the appended claims and their equivalents.

Claims

1. A pharmaceutical composition comprising an outer shell, the outer shell comprising a membrane formed by fusion of a first membrane component and a second membrane component, the first membrane component comprising a membrane derived from an inner membrane of bacteria, the bacteria being selected from the genera of the following group: Escherichia coli, Staphylococcus, Bacillus, Lactobacillus, Klebsiella, Brucella, Proteus, Acinetobacter and Pseudomonas, and any combination thereof, the second membrane component comprising a membrane derived from the cell membrane of another organism, the other organism being a tumor cell selected from the group consisting of breast cancer cells, colon cancer cells, liver cancer cells, gastric cancer cells, renal cancer cells, pancreatic cancer cells, ovarian cancer cells, lymphoma cells, osteosarcoma cells, glioma cells, prostate cancer cells and melanoma cells, and any combination thereof, the mass ratio of the first membrane component to the second membrane component in the pharmaceutical composition being 1:75 to 100:1, and the pharmaceutical composition further comprising an inner core comprising a biocompatible material.

2. The pharmaceutical combination according to claim 1, wherein the components of the other organisms comprise immunogenic components.

3. The pharmaceutical combination of claim 1, wherein the component of the other organism is capable of eliciting an immune response against the organism. The pharmaceutical combination according to claim 1 , wherein the component of the other organism comprises a tumor antigen or a functionally active fragment thereof.

5. The pharmaceutical combination according to claim 1, wherein the component of the other organism comprises a protein or a functionally active fragment thereof selected from the group consisting of antigen peptide transporter 1, H-2 class II histocompatibility antigen γ chain, tyrosine protein kinase SYK, high affinity immunoglobulin epsilon receptor subunit γ, Ras-related C3 botulinum toxin substrate 2, tyrosine protein kinase BTK, receptor-type tyrosine protein phosphatase C, Na + / K + -ATPase, ATP5A (IM CVa), ubiquinone cytochrome c reductase core protein I (IMCore I), VDAC1 / porin (OM Porin), matrix cyclophilin D (Matrix CypD), intermembrane space cytochrome c (IMSCytc), basement membrane-specific heparan sulfate proteoglycan core protein (HSPG2), plasma membrane calcium-transporting ATPase 1 (ATP2b1), endoplasmic reticulum membrane protein complex subunit 1 (EMC1), transmembrane protein 43 (TMEM43), vesicle membrane protein VIP36 (LMAN2), vesicle-associated membrane protein-associated protein A (VAPA), transmembrane 9 superfamily member 2 (TM9SF2), transmembrane emp24 domain-containing protein 10 (TMED10), adipocyte plasma membrane-associated protein (APAMP), synaptic vesicle membrane protein VAT (VAT1), nuclear pore membrane glycoprotein 210 (NUP210), plasma membrane calcium-transporting ATPase 4 (ATP2b4), plasma membrane calcium-transporting ATPase 2 (ATP2b2), erythrocyte band 7 integral membrane protein (STOM), transmembrane emp24 domain-containing protein 9 (TMED9), mitochondrial import inner membrane transposase subunit TIM44 (TIMM44), membrane-associated progesterone receptor component 1 (PGRMC1), ER membrane protein complex subunit 3 (EMC3), vesicle-associated membrane protein-associated protein B (VAPB), membrane primary amine oxidase (AOC3), vesicle-associated membrane protein 7 (VAMP7), Golgi membrane protein 4 (GOLIM4), membrane-associated progesterone receptor component 2 (PGRMC2), transmembrane 9 superfamily member 3 (TM9SF3), transmembrane 9 superfamily member 4 (TM9SF4), endoplasmic reticulum membrane protein complex subunit 2 (EMC2), mitochondrial import inner membrane transposase subunit T IM50 (TIMM50), peroxisomal membrane protein 11B (PEX11b), transmembrane emp24 domain-containing protein 2 (TMED2), secretory carrier-associated membrane protein 3 (SCAMP3), thioredoxin-related transmembrane protein 4 (TMX4), peroxisomal membrane protein PMP34 (SLC25a17), peroxisomal membrane protein PEX14 (PEX14), endoplasmic reticulum membrane protein complex subunit 8 (EMC8), interferon-induced transmembrane protein 3 (IFITM3), lysosome-associated membrane glycoprotein 2 (LAMP2), thioredoxin-related transmembrane protein 2 (TMX2), vesicle-associated membrane protein 3 (VAMP3), lysosome-associated membrane glycoprotein 1 (LAMP1), mitochondrial import inner membrane transposase subunit Tim8 A (TIMM8a1), lysosomal membrane protein 2 (SCARB2), Iggamma-2A chain C region (IGHG2a), transmembrane 9 superfamily member 1 (TM9SF1), inner nuclear membrane protein Man1 (LEMD3), transmembrane emp24 domain-containing protein 4 (TMED4), Thy-1 membrane glycoprotein (Thy1), mitochondrial import inner membrane transposase subunit Tim23 (TIMM23), mitochondrial import inner membrane transposase subunit Tim9 (TIMM9), mitochondrial import inner membrane transposase subunit Tim13 (TIMM13), secretory carrier-associated membrane protein 2 (SCAMP2), secretory carrier-associated membrane protein 1 (SCAMP1), integral membrane protein 2B (ITM2b), mitochondrial import inner membrane transposase subunit Ti m10 (TIMM10), vacuole membrane protein 1 (VMP1), growth hormone-induced transmembrane protein (GHITM), death domain-containing membrane protein (NRADD), vesicle-associated membrane protein 8 (VAMP8), transmembrane anterior-posterior transition protein 1 (TAPT1), mitochondrial intermembrane space import and assembly protein 40 (CHCHD4), glycosylated lysosomal membrane protein (GLMP), transmembrane 4 domain superfamily A member 6D (MS4A6D), translocation chain-associated membrane protein 1 (TRAM1), interferon-induced transmembrane protein 2 (IFITM2), sarcolemma-associated protein (SLMAP), membrane magnesium transporter 1 (MMGT1), nuclear envelope pore membrane protein POM121 (POM121), transmembrane protein C16orf54 homologous protein (AI467606), vacuolar ATPase assembly integral membrane protein Vma21 (VMA21), epithelial membrane protein 1 (EMP1), membrane-associated phosphatidylinositol transfer protein 1 (PITPNM1), small integral membrane protein 15 (SMIM15), nuclear integral membrane protein 1 (NEMP1), integral membrane protein GPR180 (GPR180), secretory carrier-associated membrane protein 4 (SCAMP4), translocation chain-associated membrane protein 2 (TRAM2), transmembrane and coiled-coil domain protein 3 (TMCC3), matrix membrane-associated protein 1 (SMAP1), neuronal membrane glycoprotein M6-b (GPM6b), epithelial membrane protein 2 (EMP2), Golgi membrane protein 1 (GOLM1), SI D1 transmembrane family member 2 (SIDT2), trans-Golgi network integral membrane protein 2 (TGOLN2), Golgi membrane protein TVP23 homolog B (FAM18b), mitochondrial inner membrane protein OXA1L (OXA1L), ossification-related transmembrane protein 1 (OSTM1), peroxisomal membrane protein PEX13 (PEX13), unilaterally passing membrane and coiled-coil domain protein 1 (SMCO1), distal membrane arm assembly complex protein 1 (DMAC1), secretory carrier-associated membrane protein 5 (Scamp5), cystic fibrosis transmembrane conductance regulator (CFTR), osteoclast-stimulating transmembrane protein (OCSTAMP), fat storage-inducing transmembrane protein 2 (FITM2) and transmembrane channel-like protein 5 (TMC5), and any combination thereof.

6. The pharmaceutical combination according to claim 1, wherein the component of the other organism comprises a protein or a functionally active fragment thereof selected from the group consisting of antigen peptide transporter 1, H-2 class II histocompatibility antigen γ chain, tyrosine protein kinase SYK, high affinity immunoglobulin epsilon receptor subunit γ, Ras-related C3 botulinum toxin substrate 2, tyrosine protein kinase BTK, receptor-type tyrosine protein phosphatase C, Na + / K + -ATPase, ATP5A (IM CVa), ubiquinone cytochrome c reductase core protein I (IMCore I), VDAC1 / porin (OM Porin), matrix cyclophilin D (Matrix CypD) and intermembrane space cytochrome c (IMSCytc), and any combination thereof.

7. The pharmaceutical combination according to claim 1, wherein the inner membrane of the bacterium comprises a protein or a functionally active fragment thereof selected from the group consisting of cell division protein FtsZ, inner membrane protein YhcB, inner membrane protein translocation enzyme YidC, cell division protein NlpI, ABC transporter MsbA, inner membrane transport protein TatA, intermembrane phospholipid transport system lipoprotein MlaA, inner membrane protein TolQ, intermembrane transport lipoprotein PqiC, outer membrane protein TolC, outer membrane introduction protein FimD, outer membrane porin OmpC, intermembrane transport protein PqiB, major outer membrane lipoprotein Lpp, membrane-bound hemolytic wall protein transglycosylase MltB, UPF0194 membrane protein YbhG, putative membrane protein IgaA homolog, transmembrane phospholipid transport system lipoprotein MlaA, inner membrane protein YejM, membrane-bound hemolytic wall protein transglycosylase MltA, transmembrane phospholipid transport system binding protein MlaC, inner membrane protein YlaC, transmembrane transport protein YebT, membrane-bound hemolytic murein transglycosylase MltC, transmembrane phospholipid transport system ATP-binding protein MlaF, intermembrane phospholipid transport system binding protein MlaD, inner membrane protein Membrane protein YqjE, UPF0053 inner membrane protein YfjD, UPF0053 inner membrane protein YoaE, inner membrane type cytolytic murein transglycosylase A, UPF0394 inner membrane protein YedE, intermembrane phospholipid transport system binding protein MlaB, inner membrane protein YccF, transmembrane phospholipid transport system permease protein MalE, inner membrane protein YedI, inner membrane protein YgaP, intermembrane transport protein PqiA, UPF0056 membrane protein YhcE, inner membrane protein YbhL, inner membrane protein YhjD, inner membrane transport protein YbaT, inner membrane protein White YjjP, inner membrane protein YhaH, inner membrane protein YbjJ, inner membrane transport protein YqeG, UPF0053 inner membrane protein YtfL, arsenic pump membrane protein ArsB, inner membrane protein YpjD, membrane-bound lysozyme inhibitor MliC of C-type lysozyme, UPF0283 membrane protein YcjF, UPF0259 membrane protein YciC, inner membrane protein YgfX, inner membrane protein YbbJ, lipoprotein release system transmembrane protein LolE, inner membrane protein YjcH, protein export membrane protein SecG, inner membrane protein YfdC, UPF0324 inner membrane protein YeiH, UPF0266 membrane protein YobD, TVP38 / TMEM64 family inner membrane protein YdjZ, membrane-associated protein UidC, inner membrane protein YbiR, inner membrane protein YhiM, membrane transport protein YfcA, inner membrane protein YdgK, membrane-bound hemolytic wall protein transglycosylase F, multidrug resistance outer membrane protein MdtP, UPF0208 membrane protein YfbV, peptidoglycan-associated lipoprotein, lipoprotein YiaD, lipoprotein YeaY, apolipoprotein N-acyltransferase, D-methionine-binding lipoprotein MetQ, lipoprotein GfcD, lipoprotein YbjP, Lipoprotein YgeR, lipoprotein release system ATP-binding protein LolD, osmotic-inducible lipoprotein OsmE, osmotic-inducible lipoprotein OsmB, lipoprotein YdcL, lipoprotein YajG, lipoprotein NlpE, phosphatidylglycerol-lipoprotein diacylglycerol transferase, lipoprotein YghJ, lipoprotein YedD, lipoprotein YifL, lipoprotein YgdI, lipoprotein YbaY, lipoprotein signal peptidase, lipoprotein YceB, lipoprotein YajI and inner membrane protein YebE, and any combination thereof.

8. The pharmaceutical combination according to claim 1, wherein the bacterial inner membrane comprises a protein or a functionally active fragment thereof selected from the group consisting of FtsZ, YhcB, YidC, NlpI, MsbA, TatA, MlaA, TolQ and YebE, and any combination thereof.

9. The pharmaceutical composition of claim 1, wherein the core comprises a synthetic material.

10. The pharmaceutical combination according to claim 1, wherein the core comprises a substance selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), metal-organic framework (MOF) and mesoporous silica nanoparticles.

11. The pharmaceutical combination of claim 1, wherein the inner core comprises a substance selected from the group consisting of an immune adjuvant, an immune checkpoint inhibitor, a nucleic acid molecule, and a chemotherapeutic agent, and any combination thereof.

12. The pharmaceutical combination of claim 11, wherein the inner core comprises the immune adjuvant monophosphoryl lipid A.

13. The drug combination according to claim 1, wherein the core comprises doxorubicin, paclitaxel, docetaxel, gemcitabine, capecitabine, cyclophosphamide, fluorouracil, pemetrexed, raltitrexed, bleomycin, daunorubicin, doxorubicin, vincristine and etoposide, and any combination thereof.

14. The pharmaceutical combination of claim 1, wherein the inner core comprises an indoleamine 2,3-dioxygenase (IDO) inhibitor.

15. The pharmaceutical combination of claim 1, wherein the inner core comprises small interfering RNA (siRNA). The pharmaceutical combination according to claim 1 , wherein the diameter of the core is 60 to 100 nanometers.

17. The pharmaceutical combination of claim 1, wherein the inner core has a diameter of about 86 nanometers.

18. The pharmaceutical combination of claim 1, wherein the shell has a thickness of 10 to 20 nanometers.

19. The pharmaceutical combination of claim 1, wherein the shell has a thickness of about 14 nanometers.

20. The pharmaceutical combination of claim 1, wherein the outer shell has a diameter of about 100 nanometers.

21. The pharmaceutical combination of claim 1, wherein the surface potential of the shell is between +50 mV and -50 mV.

22. The pharmaceutical combination of claim 1, wherein the shell has a zeta potential of +50 mV to -50 mV.

23. The pharmaceutical combination of claim 1, wherein the surface potential of the shell is about -21 mV.

24. The pharmaceutical combination of claim 1, wherein the shell has a zeta potential of about -21 mV.

25. The pharmaceutical combination of claim 1, wherein the mass ratio of the first membrane component to the second membrane component in the pharmaceutical combination is 1:75, 1:50, 1:25, 1:10, 1:5, 1:3, 1:1, 1:0, 0:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1 or 100:

1.

26. The pharmaceutical combination according to claim 1, wherein the presence and / or ratio of the first membrane fraction and the second membrane fraction in the pharmaceutical combination is confirmed by Western blotting and / or immunogold staining.

27. The pharmaceutical combination of claim 1, comprising particles comprising the inner core and the outer shell.

28. The pharmaceutical combination of claim 27, wherein the mass ratio of the shell to the core material is 1:1 to 1:

10.

29. The pharmaceutical combination of claim 27, wherein the mass ratio of the shell to the core material is 1:4 to 1:

6.

30. The pharmaceutical combination of claim 27, wherein the lipopolysaccharide (LPS) content in the particles is not significantly different from the LPS content of mammalian cells.

31. The pharmaceutical composition of claim 27, wherein the particles have a diameter of 70 to 120 nanometers.

32. The pharmaceutical combination of claim 27, wherein the particles have a diameter of about 100 nanometers.

33. The pharmaceutical combination of claim 31 or 32, wherein the diameter is measured by transmission electron microscopy (TEM).

34. The pharmaceutical composition of claim 27, wherein the hydrated particle size of the particles is 150 nm to 250 nm.

35. The pharmaceutical composition of claim 27, wherein the particles have a hydrated particle size of about 180 nanometers.

36. The pharmaceutical combination of claim 27, wherein the particles have a surface potential of +50 mV to -50 mV.

37. The pharmaceutical combination of claim 27, wherein the particles have a zeta potential of +50 mV to -50 mV.

38. The pharmaceutical combination of claim 27, wherein the particles have a surface potential of about -21 mV.

39. The pharmaceutical combination of claim 27, wherein the particles have a zeta potential of about -21 mV.

40. The pharmaceutical combination according to claim 27, wherein the hydrated particle size and / or surface potential of the particles are measured by a dynamic light scattering (DLS) instrument.

41. The pharmaceutical combination of claim 27, wherein the particles are capable of maintaining stability in solution.

42. The pharmaceutical combination of claim 41, wherein the stability comprises that the hydrated particle size and / or surface potential of the particles after being stored for a period of time are not significantly different from those before the period of time.

43. The pharmaceutical combination of claim 41, wherein the stability comprises that the hydrated particle size and / or surface potential of the particles after being stored in phosphate buffered saline (PBS) at 4 degrees Celsius for a period of time are not significantly different from those before the period of time.

44. The pharmaceutical combination of claim 42 or 43, wherein the period of time is not less than 21 days.

45. The pharmaceutical combination of claim 1, further comprising a substance selected from the group consisting of an immune adjuvant, an immune checkpoint inhibitor, a nucleic acid molecule, a chemotherapeutic agent, and a photosensitizer, and any combination thereof.

46. ​​The pharmaceutical combination of claim 45, further comprising the immune adjuvant monophosphoryl lipid A (MPLA).

47. The pharmaceutical combination of claim 1, further comprising an indoleamine 2,3-dioxygenase (IDO) inhibitor.

48. The pharmaceutical combination of claim 1, further comprising small interfering RNA (siRNA).

49. A vaccine comprising the pharmaceutical combination according to any one of claims 1 to 48.

50. A kit comprising the pharmaceutical combination according to any one of claims 1 to 48 and / or the vaccine according to claim 49.

51. Use of the drug combination according to any one of claims 1 to 48, the vaccine according to claim 49 and / or the kit according to claim 50 in the preparation of a medicament for preventing and / or treating a tumor, wherein the tumor is selected from the following group: breast tumor, colon tumor, liver tumor, gastric tumor, kidney tumor, pancreatic tumor, ovarian tumor, lymphoma, osteosarcoma, glioma, prostate cancer and melanoma, and any combination thereof.

52. The use according to claim 51, wherein the prevention and / or treatment of tumors comprises slowing down the rate of increase in tumor volume and / or reducing the volume of the tumor. The use according to claim 51 , wherein the tumor comprises a tumor that was not completely removed after tumor resection. The use according to claim 51 , wherein the tumor comprises a tumor that re-emerged after the tumor was eliminated.

55. The use according to claim 51, wherein the medicament is used to enhance innate immunity and / or specific immune response. The use according to claim 51 , wherein administration of the drug combination promotes dendritic cell maturation and / or increases cytokine secretion by lymphocytes compared with no administration of the drug combination.

57. The use according to claim 51, wherein the use does not substantially induce a systemic inflammatory response.

58. The use of claim 51, wherein administration of the drug combination does not substantially increase the concentration of IFN-γ, TNF-α, macrophage chemoattractant protein-1 (MCP-1), IL-12p70, IL-10, IL-23, IL-27, IL-17A, IFN-β, granulocyte-macrophage colony-stimulating factor (GM-CSF) and / or IL-1α in the serum of the subject compared to not administering the drug combination.

59. The use according to claim 51, wherein the use does not substantially induce hemolytic reaction, heart damage, liver damage, spleen damage, lung damage and / or kidney damage.

60. A method for preparing the pharmaceutical combination according to any one of claims 1 to 48, the vaccine according to claim 49 and / or the kit according to claim 50, comprising providing the membrane derived from the inner membrane of a bacterium.

61. The method of claim 60, further comprising providing said component derived from an organism other than said bacterium.

62. The method of claim 61, further comprising mixing the membrane derived from the inner membrane of bacteria and the components derived from an organism other than the bacteria to provide an outer shell.

63. The method of claim 60, further comprising providing a kernel.

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